Carbonyl reduction of bupropion in human liver

Bupropion is metabolized extensively in humans by oxidative and reductive processes. CYP2B6 mediates oxidation of bupropion to hydroxybupropion, but the enzyme(s) catalyzing carbonyl reduction of bupropion to erythro- and threohydrobupropion in human liver is unknown. The objective of this study was to examine the enzyme kinetics of bupropion reduction in human liver. In human liver cytosol, the reduction of bupropion to erythro-and threohydrobupropion was NADPH dependent with Cl(int) values of 0.08 and 0.60 μL·min(-1)mg(-1) protein, respectively. Bupropion reduction in liver microsomes was also NADPH dependent with Cl(int) values of 10.4 and 280 μL·min(-1)mg(-1) protein, respectively. Formation of erythro-and threohydrobupropion in microsomes exceeded that in cytosol by 70 and 170 fold, respectively. Menadione, an inhibitor of cytosolic carbonyl reducing enzymes (e.g. CBRs), inhibited erythro-and threohydrobupropion formation in cytosol with IC(50) of 30 and 54 μM, respectively. In microsomes 18β-glycyrrhetinic acid, an inhibitor of microsomal carbonyl reductases (e.g. 11β-HSDs), inhibited their formation with IC(50) of 25 and 26?nM, respectively. Our findings, in agreement with recent human placental studies, show that carbonyl reducing enzymes in hepatic microsomes are significant players in bupropion reduction. Contrary to past studies, we found that threohydrobupropion (not hydroxybupropion) is the major microsomal generated hepatic metabolite of bupropion.     

Effect of St. John’s wort supplementation on the pharmacokinetics of bupropion in healthy male Chinese volunteers

1. The objective of this study was to investigate the effects of continuous St. John’s wort administration on single-dose pharmacokinetics of bupropion, a substrate of cytochrome P450 (CYP) 2B6, in healthy Chinese volunteers.

2. Eighteen unrelated healthy male subjects participated in this study. The single-dose pharmacokinetics of bupropion and hydroxybupropion were determined before (control) and after a long-term period of St. John’s wort intake (325?mg, three times a day for 14 days). Plasma concentrations of bupropion and hydroxybupropion were determined before and at 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 24, 36, 48, 60 and 72?h after dosing.

3. St. John’s wort treatment decreased the area under the concentration versus time curve extrapolated to infinity of bupropion in healthy volunteers from 1.4 μg·h ml^?1 (95% confidence interval [CI]?=?1.2–1.6 μg·h ml^?1) after bupropion alone to 1.2 μg·h ml^?1 (95% CI?=?1.1–1.3 μg·h ml^?1) during St. John’s wort treatment. St. John’s wort treatment increased the oral clearance of bupropion from 108.3 l h^?1 (95% CI?=?95.4–123.0 l h^?1) to 130.0 l h^?1 (95% CI?=?118.4–142.7 l h^?1). No change in the time to peak concentration (t_max) and the blood elimination half-life (t_1/2) of bupropion was observed between the control and St. John’s wort-treated phases. However, the half-life of hydroxybupropion between two phases had a significant difference by a Student’s t test after logarithmic transformation. St. John’s wort treatment decreased the half-life of hydroxybupropion from 26.7?h (95% CI?=?23.8–29.9?h) to 24.4?h (95% CI?=?21.9–27.3?h).

4. St. John’s wort decreased, to a statistically significant extent, the plasma concentrations of bupropion, probably mainly by increasing the clearance of bupropion.

     

The in vitro metabolism of bupropion revisited: concentration dependent involvement of cytochrome P450 2C19

1. The involvement of cytochrome P450 2B6 (CYP2B6) to the in vitro and in vivo metabolism of bupropion has been well studied. In these investigations we performed a detailed in vitro phenotyping study to characterize isoforms other than CYP2B6.

2. A total of nine metabolites were identified (M1–M9) in the incubations with cDNA-expressed P450s (rhCYP) and human liver microsomes (HLM).

3. Incubations in rhCYP identified CYP2B6 as the isoform responsible for the formation of hydroxybupropion (M3). CYP2C19 was involved in bupropion metabolism primarily through alternate hydroxylation pathways (M4–M6) with higher activity at lower substrate concentrations, near 1 µM.

4. The results from HLM inhibition studies using CYP2B6 and CYP2C19 inhibitory antibodies indicated that CYP2B6 contributed to approximately 90% of M3 formation, and CYP2C19 contributed to approximately 70–90% of M4, M5, and M6 formation.

5. Studies using single donor HLM with varying degrees of CYP2B6 and CYP2C19 activities showed a good relationship between M3 formation and CYP2B6 activity and M4/M5 formation and CYP2C19 activity.

6. These results confirmed the principle role of CYP2B6 in hydroxybupropion formation, as a selective CYP2B6 probe. In addition, the new findings revealed that CYP2C19 also contributes to bupropion metabolism through alternate hydroxylation pathways.

     

Role of individual human cytochrome P450 enzymes in the in vitro metabolism of hydromorphone

1. The aim was to identify the individual human cytochrome P450 (CYP) enzymes responsible for the in vitro N-demethylation of hydromorphone and to determine the potential effect of the inhibition of this metabolic pathway on the formation of other hydromorphone metabolites.

2. Hydromorphone was metabolized to norhydromorphone (apparent K_m = 206?? 822?μM, V_max = 104 ? 834?pmol?min^?1?mg^?1 protein) and dihydroisomorphine (apparent K_m = 62 ? 557?μM, V_max = 17 ? 122?pmol?min^?1?mg^?1 protein) by human liver microsomes.

3. In pooled human liver microsomes, troleandomycin, ketoconazole and sulfaphenazole reduced norhydromorphone formation by an average of 45, 50 and 25%, respectively, whereas furafylline, quinidine and omeprazole had no effect. In an individual liver microsome sample with a high CYP3A protein content, troleandomycin and ketoconazole inhibited norhydromorphone formation by 80%.

4. The reduction in norhydromorphone formation by troleandomycin and ketoconazole was accompanied by a stimulation in dihydroisomorphine production.

5. Recombinant CYP3A4, CYP3A5, CYP2C9 and CYP2D6, but not CYP1A2, catalysed norhydromorphone formation, whereas none of these enzymes was active in dihydroisomorphine formation.

6. In summary, CYP3A and, to a lesser extent, CYP2C9 catalysed hydromorphone N-demethylation in human liver microsomes. The inhibition of norhydromorphone formation by troleandomycin and ketoconazole resulted in a stimulation of microsomal dihydroisomorphine formation.

     

Stereoselective degradation of metalaxyl and its enantiomers in rat and rabbit hepatic microsomes in vitro

1. The stereoselective degradations of racemate metalaxyl (rac-MX) and its single enantiomers in rat and rabbit hepatic microsomes were assayed by a chiral high-performance liquid chromatography method.

2. The t_1/2 of (+)-S-MX in rat liver microsomes was between 7–8?min tested by rac-MX and the individual (+)-S-enantiomer, respectively, and that for (?)-R-MX was 15–16?min. In contrast, t_1/2 in rabbit liver microsomes was much longer and showed great difference when using racemate and single enantiomer, which was similar to the results of in vivo study.

3. The enantioselectivity in rat hepatic microsomes was more evident and the degradations of MX enantiomers in rat and rabbit hepatic microsomes were Nicotinamide adenine dinucleotide phosphate-dependent.

4. Michaelis constant (K_m) and intrinsic metabolic clearance (CL_int) of (+)-S-MX were larger than that of (?)-R-MX and there was no chiral inversion from (+)-S-MX to (?)-R-MX or vice versa in both rat and rabbit hepatic microsomes.

     

Comparison of intrinsic metabolic clearance in fresh and cryopreserved human hepatocytes

1. We compared the intrinsic clearance (CL_int) of a number of substrates in suspensions of fresh and cryopreserved human hepatocytes from seven donors.

2. CL_int values for a cocktail incubation of phenacetin, diclofenac, diazepam, bufuralol, midazolam, and hydroxycoumarin were 4.9?±?3.4, 18?±?7.2, 5.1?±?4.9, 6.3?±?3.3, 9.8?±?5.8 and 22?±?14?μl min^?1/10⁶ cells, respectively, and they correlated well with corresponding CL_int values using cryopreserved hepatocytes from 25 different donors.

3. CL_int values of each cocktail substrate and 20 AstraZeneca new chemical entities were compared in fresh and cryopreserved hepatocytes from the same three donors. There was a statistically significant correlation between CL_int in fresh and cryopreserved hepatocytes for each of the three livers (p?int values was 1.03.

4. In conclusion, the results add further support to the use of cryopreserved human hepatocytes as a screening model for the intrinsic clearance of new chemical entities.

     

Use of uptake intrinsic clearance from attached rat hepatocytes to predict hepatic clearance for poorly permeable compounds

1. We previously reported that the accuracy of clearance (CL) prediction could be differentiated by permeability. CL was drastically under-predicted by in vitro metabolic intrinsic clearance (CL_int) for compounds with low permeability (<5?×?10^?6 cm/s).

2. We determined apparent uptake CL_int by measuring initial disappearance from medium using attached rat hepatocytes and metabolic CL_int by measuring parent depletion in suspended rat hepatocytes (cells and medium).

3. Uptake and metabolic CL_int were comparable for highly permeable metabolic marker compounds. In contrast, uptake CL_int was 3- to 40-fold higher than metabolic CL_int for rosuvastatin, bosentan, and 15 proprietary compounds, which had low permeability, suggesting that uptake could be a rate-determining step in hepatic elimination for these poorly permeable compounds.

4. The prediction of hepatic CL was improved significantly when using uptake CL_int for the compounds with low permeability. The average fold error was 2.2 and 6, as opposed to >11 and >47 by metabolic CL_int, with and without applying a scaling factor of 4, respectively.

5. Uptake CL_int from attached hepatocytes can be used as an alternative approach to predict hepatic clearance and to understand the significance of hepatic uptake in elimination in an early drug discovery setting.

     

Metabolism of bupropion by baboon hepatic and placental microsomes

The aim of this investigation was to determine the biotransformation of bupropion by baboon hepatic and placental microsomes, identify the enzyme(s) catalyzing the reaction(s) and determine its kinetics. Bupropion was metabolized by baboon hepatic and placental microsomes to hydroxybupropion (OH-BUP), threo- (TB) and erythrohydrobupropion (EB). OH-bupropion was the major metabolite formed by hepatic microsomes (K_m 36 ± 6 μM, V_max 258 ± 32 pmol mg protein⁻¹ min⁻¹), however the formation of OH-BUP by placental microsomes was below the limit of quantification. The apparent K_m values of bupropion for the formation of TB and EB by hepatic and placental microsomes were similar. The selective inhibitors of CYP2B6 (ticlopidine and phencyclidine) and monoclonal antibodies raised against human CYP2B6 isozyme caused 80% inhibition of OH-BUP formation by baboon hepatic microsomes. The chemical inhibitors of aldo-keto reductases (flufenamic acid), carbonyl reductases (menadione), and 11β-hydroxysteroid dehydrogenases (18β-glycyrrhetinic acid) significantly decreased the formation of TB and EB by hepatic and placental microsomes. Data indicate that CYP2B of baboon hepatic microsomes is responsible for biotransformation of bupropion to OH-BUP, while hepatic and placental short chain dehydrogenases/reductases and to a lesser extent aldo-keto reductases are responsible for the reduction of bupropion to TB and EB.     

Influence of carboxylesterase 2 genetic polymorphisms on mycophenolic acid pharmacokinetics in Japanese renal transplant recipients

1. Mycophenolic acid (MPA), converted from the prodrug mycophenolate mofetil (MMF), is generated by intestinal and hepatic esterases. The role of carboxylesterase (CES) in MMF hydrolysis was examined in vitro using human liver microsomes. V_max and K_m values of MMF hydrolysis in pooled human liver microsomes were 1368?±?44 nmol min^?1 mg^?1 protein and 1030?±?65?μM, respectively.

2. Hydrolytic activity was inhibited by the CES inhibitors phenylmethylsulfonylfluoride, bis-p-nitorophenylphosphate and diisopropylfluorophosphate, with IC₅₀ values of 77.1, 3.59 and 0.0312?μM, respectively.

3. Eighty Japanese renal transplant recipients that received repeated-doses of MMF, tacrolimus and prednisolone, were evaluated for MPA pharmacokinetics 28 days after transplantation to investigate the relationship between MPA pharmacokinetics and CES2 genetic polymorphisms.

4. No significant differences in MPA pharmacokinetics were observed between CES2 A4595G, C8721T or A-1548G genotype groups. CES2 allelic variants also did not appear to affect plasma MPA concentrations between individuals.

5. In conclusion, the study demonstrated that while CES1 and/or CES2 are involved in the hydrolysis of MMF to MPA, CES2 allelic variants appeared to make only a minor contribution to inter-personal differences in MPA pharmacokinetics.

     

CYP1A2 is the major isoform responsible for paeonol O-demethylation in human liver microsomes

1. Paeonol, the primary active component of a traditional Chinese medicine Moutan Cortex, has a wide range of pharmacological activities. In the present study, the metabolism of paeonol by cytochrome P450s (CYPs) was investigated in human liver microsomes.

2. One O-demethylated metabolite was detected in reaction catalysed by human liver microsomes, and was identified as resacetophenone by comparing the tandem mass spectra and the chromatographic retention time with that of the standard compound.

3. The study with a chemical selective inhibitor, cDNA-expressed human CYPs, a correlation assay, and a kinetics study demonstrated that CYP1A2 was the major isoform responsible for the paeonol O-demethylation in human liver microsomes.

     

Assessment of the in vitro cytochrome P450 (CYP) inhibition potential of ZYTP1, a novel poly (ADP-ribose) polymerase inhibitor

Abstract

1. ZYTP1 is a novel Poly (ADP-ribose) polymerase protein inhibitor being developed for cancer indications.

2. The focus of the work was to determine if ZYTP1 had a perpetrator role in the in vitro inhibition of cytochrome P450 (CYP) enzymes to aid dosing decisions during the clinical development of ZYTP1.

3. ZYTP1 IC₅₀ for CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4/5 was determined using human liver microsomes and LC-MS/MS detection. CYP3A4/5 IC₅₀ of depropylated metabolite of ZYTP1 was also determined. Time dependent inhibition of CYP3A4/5 by ZYTP1 was also assessed using substrates, testosterone and midazolam.

4. The mean IC₅₀ values of ZYTP1 were >100 µM for CYP1A2, 2B6 and 2D6, while 56.1, 24.5, 39.5 and 23.3–58.7 µM for CYP2C8, 2C9, 2C19 and 3A4/5, respectively. The CYP3A4/5 IC₅₀ of depropylated metabolite was 11.95–24.51 µM. Time dependent CYP3A4/5 inhibition was noted for testosterone and midazolam with IC₅₀ shift of 10.9- and 39.9-fold, respectively. With midazolam, the kinact and KI values of ZYTP1 were 0.075?min^?1 and 4.47 µM for the CYP3A4/5 time dependent inhibition, respectively.

5. Because of potent inhibition of CYP3A4/5, drugs that undergo metabolism via CYP3A4/5 pathway should be avoided during ZYTP1 therapy.

     

Species differences for stereoselective metabolism of ethofumesate and its enantiomers in vitro

1. The stereoselective metabolism of ethofumesate (ETO) and its enantiomers in rabbit and rat liver microsomes have been studied by chiral high-performance liquid chromatography (HPLC) method. Two metabolites were detected in both liver microsomes in the presence of β-nicotinamide adenine dinucleotide phosphate (NADPH).

2. The T_1/2 of (+)-ETO and (?)-ETO in rabbit liver microsomes were 12.2 and 4.7?min of rac-ETO and 25.9 and 6.7 of ETO enantiomers. However, the T_1/2 of (+)-ETO and (?)-ETO in rat liver microsomes were 5.3 and 5.9?min of rac-ETO and 7.8 and 10.6 of ETO enantiomers. The stereoselective selectivity is similar to the in vivo study.

3. After incubation of ETO enantiomers, stereoselectivity was present in the formation of ETO-OH enantiomer in rabbit liver microsomes, but stereoselectivity was not evident in rat liver microsomes.

4. There was no chiral inversion from the (+)-ETO to (?)-ETO or inversion from (?)-ETO to (+)-ETO in both rabbit and rat liver microsomes.

     

Effects of Escherichia coli lipopolysaccharide on the metformin pharmacokinetics in rats

1. The time-dependent (2-h, 24-h, and 96-h) effects of Escherichia coli lipopolysaccharide (ECLPS) on the intravenous (100?mg kg^?1) and oral (100?mg kg^?1) metformin pharmacokinetics were evaluated in rats.

2. After the intravenous administration of metformin to 24-h and 96-h ECLPS rats, the total area under the plasma concentration–time curve from time zero to time infinity (AUCs) and time-averaged non-renal clearances (CL_NRs) of metformin were significantly greater and slower, respectively, than the controls. However, after the oral administration of metformin, the AUCs of metformin were comparable among four groups of rats.

3. The greater (slower) intravenous AUCs (CL_NRs) of metformin in 24-h and 96-h ECLPS rats were due to the slower hepatic intrinsic clearance (CL_int) because of a decrease in the protein expression of hepatic cytochrome P450 (CYP) 2C11 and/or CYP3A subfamily than controls. The comparable oral AUCs among four groups of rats were mainly due to the comparable gastrointestinal metabolism (CL_int).

     

In vitro-in vivo correlation for intrinsic clearance for CP-409,092 and sumatriptan: a case study to predict the in vivo clearance for compounds metabolized by monoamine oxidase

1. Oxidative deamination of the GABA_A partial agonist CP-409,092 and sumatriptan represents a major metabolic pathway and seems to play an important role for the clearance of these two compounds.

2. Similar to sumatriptan, human mitochondrial incubations with deprenyl and clorgyline, probe inhibitors of monoamine oxidase B and monoamine oxidase A (MAO-B and MAO-A), respectively, showed that CP-409,092 was metabolized to a large extent by the enzyme MAO-A.

3. The metabolism of CP-409,092 and sumatriptan was therefore studied in human liver mitochondria and in vitro intrinsic clearance (CL_int) values were determined and compared to the corresponding in vivo oral clearance (CL_PO) values. The overall objective was to determine whether an in vitro-in vivo correlation (IVIVC) could be described for compounds cleared by MAO-A.

4. The intrinsic clearance, CL_int, of CP-409,092 was approximately 4-fold greater than that of sumatriptan (CL_int, values were calculated as 0.008 and 0.002?ml/mg/min for CP-409,092 and sumatriptan, respectively). A similar correlation was observed from the in vivo metabolic data where the unbound oral clearance, CL(u)_PO, values in humans were calculated as 724 and 178?ml/min/kg for CP-409,092 and sumatriptan, respectively.

5. The present work demonstrates that it is possible to predict in vivo metabolic clearance from in vitro metabolic data for drugs metabolized by the enzyme monoamine oxidase.

     

Enantioselective carbonyl reduction of eperisone in human liver microsomes

1. Eperisone, 4-ethyl-2-methyl-3-piperidinopropiophenone, is a centrally acting muscle relaxant widely used to relieve muscle stiffness and back pain. In this study, enantioselectivity for carbonyl reduction of eperisone was investigated in human liver microsomes, and the enzymes involved in the carbonyl reduction were characterised.

2. Carbonyl reduction of eperisone predominantly occurred in microsomal fractions and 11β-hydroxysteroid dehydrogenase type 1(11β-HSD 1) played a major role in this reaction as judged by selective inhibition of the activity by BVT-14225 and KR-66344. The kinetic study with (+)-S- and (?)-R-eperisone showed that the formation of the carbonyl reduced metabolite (M5) from the (?)-R-isomer was more efficient than that from the (?)-S-isomer.

3. As eperisone is a racemic compound with one chiral centre, the carbonyl reduced metabolite of eperisone (M5) may have four possible diastereoisomeric structures. Chiral separation of incubation mixtures of racemic eperisone with human liver microsome revealed that (1S, 2S)-M5 and (1R, 2R)-M5 were generated specifically from (+)-S- and (?)-R-eperisone, respectively. Selective formation of anti-diastereomers was further confirmed by incubation of individual enantiomer with microsomes.

4. Carbonyl reduction of eperisone by microsomal 11β-HSD 1 may significantly contribute to the metabolic disposition of eperisone in human and (?)-R-isomer is preferentially reduced by this enzyme.

     

CYP3A catalyses schizandrin biotransformation in human,minipig and rat liver microsomes

1. Schizandrin is recognized as the major absorbed effective constituent of Fructus schisandrae, which is extensively applied in Chinese medicinal formula. The present study aimed to profile the phase I metabolites of schizandrin and identify the cytochrome P450 (CYP) isoforms involved.

2. After schizandrin was incubated with human liver microsomes, three metabolites were isolated by high-performance liquid chromatography (HPLC) and their structures were identified to be 8(R)-hydroxyl-schizandrin, 2-demethyl-8(R)-hydroxyl-schizandrin, 3-demethyl-8(R)-hydroxyl-schizandrin, by liquid chromatography-mass spectrometry (LC-MS), ¹H-nuclear magnetic resonance (NMR), and ¹³C-NMR, respectively. A combination of correlation analysis, chemical inhibition studies, assays with recombinant CYPs, and enzyme kinetics indicated that CYP3A4 was the main hepatic isoform that cleared schizandrin. Rat and minipig liver microsomes were included when evaluating species differences, and the results showed little difference among the species.

3. In conclusion, CYP3A4 plays a major role in the biotransformation of schizandrin in human liver microsomes. Minipig and rat could be surrogate models for man in schizandrin pharmacokinetic studies. Better knowledge of schizandrin’s metabolic pathway could provide the vital information for understanding the pharmacokinetic behaviours of schizandrin contained in Chinese medicinal formula.

     

The effectiveness of oracin in enhancing the cytotoxicity of doxorubicin through the inhibition of doxorubicin deactivation in breast cancer MCF7 cells

1. The maximal therapeutic doses of the cytostatic drug doxorubicin (DOX) are strictly limited by the development of systemic toxicity, especially cardiotoxicity. The inhibition of DOX-metabolizing enzymes within cancer cells is possible strategy to improve DOX efficacy. In breast cancer cells (MCF7), DOX is effectively deactivated by carbonyl reduction. The aim of the present study was to test whether isoquinoline derivative oracin (ORC) is able to inhibit DOX reductases and to enhance DOX cytotoxic efficacy.

2. The kinetics studies of DOX reduction in MCF7 cytosolic fractions were evaluated using high-performance liquid chromatography. The cytotoxicity of DOX, ORC, and DOX+ORC combinations was assayed using cell-viability tests and caspases activities and monitored using xCELLigence System for real-time cell analysis.

3. ORC significantly inhibited DOX reduction in MCF7 cytosol. Competitive inhibition was found. The viability was significantly lower in cells treated with ORC+DOX combinations in comparison to cells treated with DOX alone. Significant enhancement of DOX cytotoxicity was achieved already with 0.5 µM ORC. DOX together with ORC was able to kill about 55% cells more than DOX alone.

4. ORC significantly increases DOX efficacy in MCF7 cells probably due to the inhibition of DOX reductases.

     

A high throughput in vitro mrp2 assay to predict in vivo biliary excretion

1. Prediction of biliary excretion is a challenge for drug discovery scientists due to the lack of in vitro assays. This study explores the possibility of establishing a simple assay to predict in vivo biliary excretion via the mrp2 transport system.

2. In vitro mrp2 activity was determined by measuring the ATP-dependent uptake of 5(6)-carboxy-2′,7′-dichlorofluorescein (CDCF) in canalicular plasma membrane vesicles (cLPM) from rat livers. The CDCF uptake was time- and concentration-dependent (K_m of 2.2?±?0.3 µM and V_max of 115?±?26 pmol/mg/min) and strongly inhibited by the mrp2 inhibitors, benzbromarone, MK-571, and cyclosporine A, with IC₅₀ values ≤ 1.1 µM.

3. Low inhibition of CDCF uptake by taurocholate (BSEP inhibitor; 57 µM) and digoxin (P-gp inhibitor; 101 µM) demonstrated assay specificity towards mrp2.

4. A highly significant correlation (r²?=?0.959) between the in vitro IC₅₀ values from the described mrp2 assay and in vivo biliary excretion in rats was observed using 10 literature compounds.

5. This study demonstrated, for the first time, that a high throughput assay could be established with the capability of predicting biliary excretion in the rat using CDCF as a substrate.

     

Chan-Yu-Bao-Yuan-Tang induces apoptosis in NSCLC and SCLC cell lines via a mitochondria-mediated pathway

1. Previous clinical studies have shown efficacy and safety of the traditional Chinese medicinal herb extract Chan-Yu-Bao-Yuan-Tang (CYBYT) in lung cancer patients.

2. The effects of CYBYT on proliferation and apoptosis in the non-small cell lung cancer cell line NCI-H460 and the small cell lung cancer cell line NCI-H446 were investigated in vitro.

3. CYBYT significantly induced antiproliferative effects of NCI-H460 and NCI-H446 cells in a concentration- and time-dependent manner. The IC₅₀ values in NCI-H460 cells were 94.37 µg/mL (24?h) and 20.89 µg/mL (48?h), whereas in NCI-H446 cells IC₅₀ values were 214.72 µg/mL (24?h) and 114.58 µg/mL (48?h). Annexin V–FITC/PI staining showed CYBYT could significantly induce apoptosis in NCI-H460 and NCI-H446 cells, and the total apoptosis rates were positively correlated with the concentration and time of CYBYT treatment. Furthermore, treatment with the broad-spectrum caspase inhibitor (z-VAD-fmk) effectively protected NCI-H460 and NCI-H446 cells against CYBYT-triggered apoptosis. The apoptotic processes involved were a marked decrease in antiapoptotic protein Bcl-2 and an increase in proapoptotic protein Bax. The release of mitochondrial cytochrome c into the cytosol was also observed, which, in turn, resulted in the activation of caspase-9 and caspase-3.

4. CYBYT exerts antiproliferative and growth inhibition effects on NCI-H460 and NCI-H446 cells through the mitochondrial caspase-dependent cell death pathway.

     

Comparative inhibition of inducible and constitutive CYPs in rat hepatic microsomes by parathion

1. In microsomal fractions, the phosphorothioate pesticide parathion inhibits cytochrome P450 (CYP) enzymes by reversible and irreversible mechanisms resulting in the long-term suppression of drug oxidation. The present study evaluated the relative susceptibilities of constitutive and inducible CYP2 and CYP3 steroid hydroxylases to inhibition by the pesticide.

2. Enzyme kinetic analysis indicated that constitutive and dexamethasone (DEX)-induced androst-4-ene-3,17-dione (AD) 6β-hydroxylations were similarly susceptible to inhibition by parathion (K_m/K_i ratios 1.5–1.6). However, preincubation of parathion with NADPH-fortified microsomes intensified the extent of inhibition of CYP3A-dependent 6β-hydroxylation. Comparison of K_m/K_i ratios indicated that 6β-hydroxylation activity in fractions from DEX-pretreated rats was about twice as susceptible as the control activity to inactivation by parathion metabolites (K_m/K_i ratio of 8.0 versus 4.0).

3. The time-dependent loss of AD 6β-hydroxylation by parathion occurred more efficiently in fractions from DEX-induced liver than in control. Thus, half-times of 1.3 and 6.1?min, respectively, were determined for the inactivation of DEX-inducible and constitutive activities. Parathion concentrations required for half-maximal inactivation were 32 and 67?μM in microsomes from DEX-induced and control rats.

4. In phenobarbital (PB)-induced fractions CYP2B1-mediated AD 16β-hydroxylation was inhibited potently in a reversible fashion by parathion (K_i?=?0.37?μM; K_m/K_i ratio about 73). Inhibition was not enhanced at parathion concentrations near the K_i by a preincubation step with NADPH.

5. In control microsomes parathion elicited a type I binding interaction with oxidized CYP (K_s?=?7.7?μM, ΔA_max?=?2.2?×?10^?2?a.u.?nmol CYP^?1; ΔA_max/K_s 2.86?×?10³?a.u. nmol?CYP^?1/mM). Ligand binding was 13- and 1.6-fold more efficient in PB and DEX microsomes, respectively.

6. These findings indicate that pretreatment of rats with enzyme-inducing drugs like DEX and PB alters the profile of CYPs and their susceptibility to inhibition by parathion. Potent reversible inhibition of CYP2B1 occurred in PB-induced fractions and DEX-inducible CYPs 3A were more susceptible to mechanism-based inactivation than the corresponding constitutive CYPs from the same subfamily.