Demethylation of radiolabelled dextromethorphan in rat microsomes and intact hepatocytes.

Liver microsomal preparations are routinely used to predict drug interactions that can occur in vivo as a result of inhibition of cytochrome P450 (CYP)-mediated metabolism. However, the concentration of free drug (substrate and inhibitor) at its intrahepatic site of action, a variable that cannot be directly measured, may be significantly different from that in microsomal incubation systems. Intact cells more closely reflect the environment to which CYP substrates and inhibitors are exposed in the liver, and it may therefore be desirable to assess the potential of a drug to cause CYP inhibition in isolated hepatocytes. The objective of this study was to compare the inhibitory potencies of a series of CYP2D inhibitors in rat liver microsomes and hepatocytes. For this, we developed an assay suitable for rapid analysis of CYP-mediated drug interactions in both systems, using radiolabelled dextromethorphan, a well-characterized probe substrate for enzymes of the CYP2D family. Dextromethorphan demethylation exhibited saturable kinetics in rat microsomes and hepatocytes, with apparent Km and Vmax values of 2.1 vs. 2.8 microM and 0.74 nM x min(-1) per mg microsomal protein vs. 0.11 nM x min(-1) per mg cellular protein, respectively. Quinine, quinidine, pyrilamine, propafenone, verapamil, ketoconazole and terfenadine inhibited dextromethorphan O-demethylation in rat liver microsomes and hepatocytes with IC50 values in the low micromolar range. Some of these compounds exhibited biphasic inhibition kinetics, indicative of interaction with more than one CYP2D isoform. Even though no important differences in inhibitory potencies were observed between the two systems, most inhibitors, including quinine and quinidine, displayed 2-3-fold lower IC50 in hepatocytes than in microsomes. The cell-associated concentrations of quinine and quinidine were found to be significantly higher than those in the extracellular medium, suggesting that intracellular accumulation may potentiate the effect of these compounds. Studies of CYP inhibition in intact hepatocytes may be warranted for compounds that concentrate in the liver as the result of cellular transport.     

紫杉醇对食蟹猴和人CYP1A2、CYP3A4及CYP2A6酶代谢的影响

目的探讨紫杉醇对食蟹猴和人肝微粒体CYP1A2、CYP2A6和CYP3A4酶活性的影响。方法采用食蟹猴和人肝脏微粒体,分别以非那西汀、睾丸酮和香豆素分别作为CYP1A2、CYP2A6、CYP3A4的底物,建立CYP1A2、CYP2A6和CYP3A4体外代谢体系。采用不同浓度的紫杉醇分别与上述3种底物共同孵育于肝微粒体代谢体系中。用HPLC法分别测定各底物的代谢产物扑热息痛、6β-羟基睾丸酮、7-羟基香豆素的产生量,计算IC50值,以评估紫杉醇对CYP1A2、CYP2A6和CYP3A4代谢的影响。结果紫杉醇对食蟹猴肝微粒体3种酶的IC50值分别为570±5.9μmol/L、140±2.9μmol/L和无影响;紫杉醇对人肝微粒体3种酶的IC50值分别为193±6.6μmol/L、253±3.6μmol/L和24±1.6μmol/L。结论紫杉醇对食蟹猴肝微粒体CYP1A2和CYP3A4活性具有一定的抑制作用,但对CYP2A6酶的活性几乎没有影响。紫杉醇对人肝微粒体CYP1A2和CYP3A4活性的抑制作用较弱,但对CYP2A6酶的活性抑制作用较强,提示临床上紫杉醇与作为上述酶底物的药物联合用药时应慎重,以避免因中西药物相互作用所导致的不良反应发生。     

Development of a novel cytochrome p450 bioaffinity detection system coupled online to gradient reversed-phase high-performance liquid chromatography

A high-resolution screening platform, coupling online affinity detection for mammalian cytochrome P450s (Cyt P450s) to gradient reversed-phase high-performance liquid chromatography (HPLC), is described. To this end, the online Cyt P450 enzyme affinity detection (EAD) system was optimized for enzyme (beta-NF-induced rat liver microsomes), probe substrate (ethoxyresorufine), and organic modifier (methanol or acetonitrile). The optimized Cyt P450 EAD system has first been evaluated in a flow injection analysis (FIA) mode with 7 known ligands of Cyt P450 1A1/1A2 (alpha-naphthoflavone, beta-naphthoflavone, ellipticine, 9-hydroxy-ellipticine, fluvoxamine, caffein, and phenacetin). Subsequently, IC50 values were online in FIA-mode determined and compared with those obtained with standardmicrosomal assay conditions. The IC50 values obtained with the online Cyt P450 EAD system agreed well with the IC50 values obtained in the standard assays. For high affinity ligands of Cyt P450 1A1/1A2, detection limits of 1 to 3 pmol injected (n=3; signal to noise [S/N]=3) were obtained. The individual inhibitory properties of ligands in mixtures of the ligands were subsequently investigated using an optimized Cyt P450 EAD system online coupled to gradient HPLC. Using the integrated online gradient HPLC Cyt P450 EAD platform, detection limits of 10 to 25 pmol injected (n=1; S/N=3) were obtained for high-affinity ligands. It is concluded that this novel screening technology offers new perspectives for rapid and sensitive screening of individual compounds in mixtures exhibiting affinity for liver microsomal Cyt P450s.     

Novel semicarbazide-derived inhibitors of human dipeptidyl peptidase I (hDPPI)

Human dipeptidyl peptidase I (hDPPI, cathepsin C, EC 3.4.14.1) is a novel putative drug target for the treatment of inflammatory diseases. Using 1 as a starting point (IC50>10 microM), we have improved potency by more than 500-fold and successfully identified novel inhibitors of DPPI via screening of a one-bead-two-compounds library of semicarbazide derivatives. Selected compounds were shown to inhibit intracellular DPPI in RBL-2H3 cells. These compounds were further characterized for adverse effects on HepG2 cells (cytotoxicity and viability) and their metabolic stability in rat liver microsomes was estimated. One of the most potent inhibitors, 8 (IC50=31+/-3 nM; Ki=45+/-2 nM, competitive inhibition), is selective for DPPI over other cysteine and serine proteases, has a half-life of 24 min in rat liver microsomes, shows approximately 50% inhibition of intracellular DPPI at 20 microM and is noncytotoxic.     

Description of a 96-well plate assay to measure cytochrome P4503A inhibition in human liver microsomes using a selective fluorescent probe

The standard method to evaluate CYP3A inhibition is to study the conversion of the specific CYP3A probe testosterone to its 6 beta-hydroxy metabolite in human liver microsomes, in the absence and presence of potential inhibitors. Quantification of the 6 beta-hydroxy metabolite is achieved by HPLC resulting in a tedious and time-consuming assay. In order to increase the P450 inhibition throughput, efforts were made to find a CYP3A probe that would produce a fluorescent metabolite. This paper reports the discovery of DFB as a potential CYP3A fluorescent probe. DFB was significantly metabolized in human microsomes (approximately 1-2 nmol/(min. mg protein)) to give the fluorescent compound DFH. The involvement of CYP3A in the metabolism of DFB was determined using multiple approaches. First, incubations conducted with microsomes made from cell lines expressing single CYPs (Gentest Supersomes) indicated that CYP3A played a major role in the metabolism of DFB. Secondly, immunoinhibition studies conducted with CYP3A antibody resulted in >95% inhibition of DFB metabolism in HLM. Thirdly, inhibition studies with specific CYP1A1, 1A2, 2C8/9, 2C19, 2D6, and 2E1 chemical inhibitors did not suppress DFB activity in HLM. However, ketoconazole, miconazole, nicardipine, and nifedipine, all known CYP3A inhibitors, completely abolished the formation of DFH in HLM. The potency of several inhibitors determined using DFB and testosterone as CYP3A probes was consistent (R = 0.98). Finally, a good agreement was obtained for the formation of DFH and production of 6 beta-hydroxytestosterone when DFB and testosterone were incubated separately with various human liver microsome preparations (R = 0.94, N = 11). In order to use DFH as a fluorescent CYP3A marker in a 96-well plate format, it was important to remove the excess of NADPH at the end of the incubation because the fluorescence of NADPH interferes with DFH detection. This was achieved by adding oxidized glutathione and glutathione reductase to convert NADPH to NADP(+) which is not fluorescent. The liquid-handling steps were fully automated in a 96-well plate format and a template was designed to generate IC(50) curves and to address potential fluorescent interferences from the test compounds. The assay was found to be reproducible (intraday variability <10% and interday variability indicated less than a 2-fold variation in the IC(50) values) and is now routinely used in our laboratory to evaluate CYP3A inhibition of NCEs.     

Inhibition of cytochrome P450 activities by oleanolic acid and ursolic acid in human liver microsomes

Oleanolic acid (OA) and ursolic acid (UA), triterpene acids having numerous pharmacological activities including anti-inflammatory, anti-cancer, and hepato-protective effects, were tested for their ability to modulate the activities of several cytochrome P450 (CYP) enzymes using human liver microsomes. OA competitively inhibited CYP1A2-catalyzed phenacetin O-deethylation and CYP3A4-catalyzed midazolam 1-hydroxylation, the major human drug metabolizing CYPs, with IC50 (Ki) values of 143.5 (74.2) microM and 78.9 (41.0) microM, respectively. UA competitively inhibited CYP2C19-catalyzed S-mephenytoin 4'-hydroxylation with an IC50 (Ki) value of 119.7 (80.3) microM. However, other CYPs tested showed no or weak inhibition by both OA and UA. The present study demonstrates that OA and UA have inhibitory effects on CYP isoforms using human liver microsomes. It is thus likely that consumption of herbal medicines containing OA or UA, or administration of OA or UA, can cause drug interactions in humans when used concomitantly with drugs that are metabolized primarily by CYP isoforms. In addition, it appears that the inhibitory effect of OA on CYP1A2 is, in part, related to its anti-inflammatory and anticancer activities.     

Synthesis and structure-activity relationships of selective norepinephrine reuptake inhibitors (sNRI) with improved pharmaceutical characteristics

The design synthesis and SAR of a series of chiral ring-constrained norepinephrine reuptake inhibitors with improved physicochemical properties is described. Typical compounds are potent (IC(50)s<10 nM), selective against the other monoamine transporters, weak CYP2D6 inhibitors (IC(50)s>1 microM) and stable to oxidation by human liver microsomes. In addition, the compounds exhibit a favorable polarity profile.     

Strain differences in the liver microsomal metabolism of the experimental anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid in mice

The experimental anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is mainly metabolised by acyl glucuronidation and to a lesser degree by 6-methyl hydroxylation. Strain differences in the maximum tolerated dose (MTD) of DMXAA in mice have been observed. The aim of this study was to compare the kinetics of DMXAA acyl glucuronidation and 6-methylhydroxylation in five various mouse strains, and correlate the in vitro metabolism data with MTD observed. In all mouse strains studied, DMXAA acyl glucuronidation and 6-methylhydroxylation in the liver microsomes followed Michaelis-Menten kinetics. Significant strain variations in the kinetic parameters (K(m), V(max) and K(m)/V(max), i.e., CL(int)) for DMXAA acyl glucuronidation and 6-methylhydroxylation in mouse liver microsomes were observed. A 2-6-fold variation was spanned across strains for K(m), V(max) and CL(int), respectively, for DMXAA glucuronidation and 6-methylhydroxylation. The rank order for total CL(int) by glucuronidation and 6-methylhydroxylation was BDF1 (1.70 ml/min per g)>wild type of mice lacking IFN-gamma receptor (0.80 ml/min per g)>nude mice (0.70 ml/min per g)>Swiss CD mice (0.56 ml/min per g)>C57Bl/6 mice (0.46 ml/min per g), with a 4-fold variation between the mouse strain of the highest and lowest CL(int). There was no significant correlation between total CL(int) and MTD (r(2)=0.88, P>0.05), but the rank order for CL(int) was consistent with that for MTD. These results suggested that there were significant strain differences in DMXAA metabolism in mouse liver microsomes and the strain-related differences in the metabolism of DMXAA did not provide an explanation for the strain differences in the MTD.     

Inhibition of human hepatic CYP isoforms by mGluR5 antagonists

Characterization of new chemical entities for their potential to produce drug-drug interactions is an important aspect of early drug discovery screening. In the present study, the potential for three metabotropic glutamate receptor antagonists to interact with recombinant human CYPs was investigated. 2-Methyl-6-(phenylethenyl) pyridine (SIB-1893), 2-methyl-6-(phenylethynyl) pyridine (MPEP) and 3-[2-methyl-1,3-thiazol-4-yl) ethynyl]-pyridine (MTEP) were moderate competitive inhibitors of recombinant human CYP1A2 (Ki, 0.5-1 microM). SIB-1893, but not MPEP or MTEP, was also a moderate competitive inhibitor of CYP1B1. MPEP and MTEP were weak inhibitors of CYP2C19. None of the three compounds tested were significant inhibitors (IC(50) values >50 microM) of CYP3A4, 2C9, 2D6, 2A6, 2B6 or 2E1. The results suggest that MTEP is a selective inhibitor of CYP1A2 and may prove to be a useful tool in studying drug-drug interactions involving this enzyme.     

Inhibition of rat liver cyclic AMP-dependent protein kinase by flavonoids.

Rat liver cyclic AMP-dependent protein kinase catalytic subunit (cAK), assayed using the synthetic peptide substrate, LRRASLG, is inhibited by a range of plant-derived flavonoids. In general, maximal inhibitory effectiveness (IC50 values 1 to 2 microM) requires 2,3-unsaturation and polyhydroxylation involving at least two of the three flavonoid rings. 3-Hydroxyflavone (IC50 value 4 microM), 3,5,7,2',4'-pentahydroxyflavone (IC50 = 10 microM) and 5,7,4'-trihydroxyflavone (IC50 = 7 microM) represent somewhat less active variations from this pattern. Flavonoid O-methylation or O-glycosylation greatly decreases inhibitory effectiveness, as does 2,3-saturation. Various flavonoid-related compounds, notably gossypol (IC50 = 10 microM), also inhibit cAK. Flavonoids and related compounds are in general much better inhibitors of cAK than of avian Ca(2+)-calmodulin-dependent myosin light chain kinase or of plant Ca(2+)-dependent protein kinase. Tricetin (IC50 = 1 microM) inhibits cAK in a fashion that is non-competitive with respect to both peptide substrate and ATP (Ki value 0.7 microM). When histone III-S is used as a substrate, inhibition of cAK requires much higher flavonoid concentrations.     

Online biochemical detection of glutathione-S-transferase P1-specific inhibitors in complex mixtures

A high-resolution screening (HRS) technology is described, which couples 2 parallel enzyme affinity detection (EAD) systems for substrates and inhibitors of rat cytosolic glutathione-S-transferases (cGSTs) and purified human GST P1 to gradient reversed-phase high-performance liquid chromatography (HPLC). The cGSTs and GST P1 EAD systems were optimized and validated first in flow injection analysis (FIA) mode, and optimized values were subsequently used for HPLC mode. The IC(50) values of 8 ligands thus obtained online agreed well with the IC(50) values obtained with microplate reader-based assays. For ethacrynic acid, an IC(50) value of 1.8 +/- 0.4 microM was obtained with the cGSTs EAD system in FIA mode and 0.8 +/- 0.6 microM in HPLC mode. For ethacrynic acid with the GST P1 EAD system, IC(50) values of 6.0 +/- 2.9 and 3.6 +/- 2.8 microM were obtained in FIA and HPLC modes, respectively. An HRS GST EAD system, consisting of both the cGSTs and the GST P1 EAD system in HPLC mode in parallel, was able to separate complex mixtures of compounds and to determine online their individual affinity for cGSTs and GST P1. Finally, a small library of GST inhibitors, synthesized by reaction of several electrophiles with glutathione (GSH), was successfully screened with the newly developed parallel HRS GST EAD system. It is concluded that the present online gradient HPLC-based HRS screening technology offers new perspectives for sensitive and simultaneous screening of general cGSTs and specific GST P1 inhibitors in mixtures.     

肉桂抑制α-葡萄糖苷酶活性成分研究

为寻找肉桂中具有抑制α-葡萄糖苷酶活性的化学成分,采用高效液相色谱结合体外抑制α-葡萄糖苷酶活性筛选模型的方法,进行活性成分的跟踪分离,并对活性化合物进行酶抑制动力学研究.结果显示,肉桂石油醚提取物(IC50=350.37 μg/mL)的活性明显高于阳性对照阿卡波糖(IC50=1028.99 μg/mL),从中分离出2个活性成分,分别鉴定为桂皮醛( IC50 =277.89 μg/mL)和肉桂酸(IC50=286.22 μg/mL).酶抑制动力学结果表明它们对α-葡萄糖苷酶的抑制类型均为非竞争性抑制,Ki值分别为178.07 μg/mL和229.43 μg/mL.     

Indole derivatives as potent inhibitors of 5-lipoxygenase: design, synthesis, biological evaluation, and molecular modeling

A series of novel indole derivatives was designed, synthesized and evaluated by cell-based assays for their inhibitory activities against 5-LOX in rat peritoneal leukocytes. Most of them (30 out of 35) showed an inhibitory potency higher than the initial screening hit 1a (IC(50)=74 microM). Selected compounds for concentration-response studies showed prominent inhibitory activities with IC(50) values ranging from 0.74 microM to 3.17 microM. Four compounds (1m, 1s, 4a, and 6a) exhibited the most potent inhibitory activity compared to that of the reference drug (Zileuton), with IC(50) values less than 1 microM. Molecular modeling studies for compounds 1a, 3a, 4a, and 6a were also presented. The excellent in vitro activities of this class of compounds may possess potential for the treatment of LT-related diseases.     

Enzyme kinetic and molecular docking studies on the metabolic interactions of 1-hydroxy-2,3,5-trimethoxy-xanthone, isolated from Halenia elliptica D. Don, with model probe substrates of human cytochrome P450 enzymes

Halenia elliptica D. Don is a Tibetan herb and medicinal preparations containing Halenia elliptica have been commonly used for the treatment of hepatitis B virus infection in China. The metabolism of 1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1) to its metabolites is mediated through cytochrome P450 enzymes. This study aimed to investigate the herb-drug interaction potential of HM-1 by studying its effects on the metabolism of model probe substrates of five major CYP450 isoforms in human liver microsomes. HM-1 showed moderate inhibitory effects on CYP1A2 (IC(50)=1.06μM) and CYP2C9 (IC(50)=3.89μM), minimal inhibition on CYP3A4 (IC(20)=11.94μM), but no inhibition on model CYP2D6 (dextromethorphan) and CYP2E1 (chlorzoxazone) probe substrates. Inhibition kinetic studies showed that the K(i) values of HM-1 on CYP1A2, CYP2C9 and CYP3A4 were 5.12μM, 2.00μM and 95.03μM, respectively. HM-1 competitively inhibited testosterone 6β-hydroxylation (CYP3A4) but displayed mixed type inhibitions for phenacetin O-deethylation (CYP1A2) and tolbutamide 4-hydroxylation (CYP2C9). Molecular docking study confirmed the inhibition modes of HM-1 on these human CYP isoforms.     

High throughput and rapid screening of marine protein hydrolysates enriched in peptides with angiotensin-I-converting enzyme inhibitory activity by capillary electrophoresis

Twelve kinds of marine protein materials, including fish, shrimp, seashell, algae and seafood wastes were selected for the hydrolysis using four different proteases. The IC(50) values for angiotensin-converting enzyme (ACE) inhibitory activity of 48 hydrolysates were rapidly determined by capillary electrophoresis (CE). The values ranged from 0.17 to 501.7mg/ml, and were affected by both the marine protein resources and the selected proteases. Hydrolysates of the lowest IC(50) values were from shrimp (Acetes chinensis), shark meat, mackerel bone, Polysiphonia urceolata and Spirulina platensis, indicating these five kinds of marine food proteins contained beneficial materials for the production of ACE inhibitory peptides by proteolysis. The hydrolysates obtained using proteases Protamex and SM98011 had lower IC(50) values, showing these two proteases were superior to others. The CE method achieved the same sensitivity as the high performance liquid chromatography (HPLC) method. However, the CE method was faster and, as a result, more economical. Therefore, CE had potential for rapid screening of marine protein hydrolysates enriched in ACE inhibitory peptides.     

2H-Thieno[3,2-e]- and [2,3-e]-1,2-thiazine-6-sulfonamide 1,1-dioxides as ocular hypotensive agents: synthesis, carbonic anhydrase inhibition and evaluation in the rabbit

Novel non-chiral 2H-thieno[3,2-e]- and [2,3-e]-1,2-thiazine-6-sulfonamide 1,1-dioxides were synthesized for evaluation as potential candidates for the treatment of glaucoma. All of the compounds prepared were potent high affinity inhibitors of human carbonic anhydrase II, Ki < 0.5 nM. Additionally, inhibition of recombinant human carbonic anhydrase IV was determined for selected compounds; these were shown to be moderate to potent inhibitors of this isozyme with IC50 values ranging from 4.25 to 73.6 nM. Of the compounds evaluated for their ability to lower intraocular pressure in naturally hypertensive Dutch-belted rabbits, 5a, 17a3, 17b1, 17b2, 17h2 and 17i1 showed significant efficacy (> 20% decrease) in this model following topical ocular administration.     

State-dependent compound inhibition of Nav1.2 sodium channels using the FLIPR Vm dye: on-target and off-target effects of diverse pharmacological agents

Voltage-gated sodium channels (NaChs) are relevant targets for pain, epilepsy, and a variety of neurological and cardiac disorders. Traditionally, it has been difficult to develop structure-activity relationships for NaCh inhibitors due to rapid channel kinetics and state-dependent compound interactions. Membrane potential (Vm) dyes in conjunction with a high-throughput fluorescence imaging plate reader (FLIPR) offer a satisfactory 1st-tier solution. Thus, the authors have developed a FLIPR Vm assay of rat Nav1.2 NaCh. Channels were opened by addition of veratridine, and Vm dye responses were measured. The IC50 values from various structural classes of compounds were compared to the resting state binding constant (Kr)and inactivated state binding constant (Ki)obtained using patch-clamp electrophysiology (EP). The FLIPR values correlated with Ki but not Kr. FLIPRIC50 values fell within 0.1-to 1.5-fold of EP Ki values, indicating that the assay generally reports use-dependent inhibition rather than resting state block. The Library of Pharmacologically Active Compounds (LOPAC, Sigma) was screened. Confirmed hits arose from diverse classes such as dopamine receptor antagonists, serotonin transport inhibitors, and kinase inhibitors. These data suggest that NaCh inhibition is inherent in a diverse set of biologically active molecules and may warrant counterscreening NaChs to avoid unwanted secondary pharmacology.     

Synthesis and biochemistry of fluorescent aromatase inhibitors

Effective aromatase inhibitors have been developed that contain aryl functionalities at the 7 alpha-position of the steroid nucleus. The exact interactions of 7 alpha-substituted androstenediones with the active site of aromatase is unknown. Fluorescent derivatives may provide a useful spectroscopic method for examining the binding of these inhibitors to the microsomal complex and purified aromatase protein. Dinitrophenyl, dansyl, and naphthyl derivatives of 7 alpha-(4'-amino)phenylthio-4-androstene-3,17-dione and androstenedione were synthesized as potential fluorescent agents. An in vitro assay with human placental microsomes was used to evaluate aromatase inhibitory properties. These fluorescent compounds were effective competitive inhibitors and have apparent Ki values ranging from 24.1 to 86.7 nM.     

Discovery and molecular docking of quinolyl-thienyl chalcones as anti-angiogenic agents targeting VEGFR-2 tyrosine kinase

Vascular endothelial growth factor Receptor-2 (VEGFR-2) kinase inhibition is one of the well established strategies to promptly tackle tumor growth by suppression of angiogenesis. In the current study, structure-based virtual screening methodology of a series of quinolyl-thienyl chalcones indicated their strong potential as VEGFR-2 kinase inhibitors. In vitro VEGFR-2 kinase inhibitory activity was found to be significant (compound 19, IC(50): 73.41nM). All compounds showed significant inhibition of human umbilical vein endothelial cells (HUVEC) proliferation (compound 19, IC(50): 21.78nM). Molecular interactions of the compounds were studied using molecular docking studies.     

Reduction of toxic metabolite formation of acetaminophen

Acetaminophen is a widely used over-the-counter drug that causes severe hepatic damage upon overdose. Cytochrome P450-dependent oxidation of acetaminophen results in the formation of the toxic N-acetyl-p-benzoquinone-imine (NAPQI). Inhibition of cytochrome P450 enzymes responsible for NAPQI formation might be useful--besides N-acetylcysteine treatment--in managing acetaminophen overdose. Investigations were carried out using human liver microsomes to test whether selective inhibition of cytochrome P450s reduces NAPQI formation. Selective inhibition of CYP3A4 and CYP1A2 did not reduce, whereas the inhibition of CYP2A6 and CYP2E1 significantly decreased NAPQI formation. Furthermore, selective CYP2E1 inhibitors that are used in human therapy were tested for their inhibitory effect on NAPQI formation. 4-Methylpyrazole, disulfiram, and diethyl-dithiocarbamate were the most potent inhibitors with IC(50) values of 50 microM, 8 microM, and 33 microM, respectively. Although cimetidin is used in the therapy of acetaminophen overdose as an inhibitor of cytochrome P450, it is not able to reduce NAPQI formation.