Inhibition of CYP3A4 and CYP2C9 by podophyllotoxin: Implication for clinical drugdrug interactions

JIN-HUI SONG1 , DONG-XUE SUN2 , BIN CHEN3 , DAI-HONG JI3 , JIE PU3 , JIE XU1 , FENG-DE TIAN1 and LIN GUO1,*
1

Orthopedics Department 2, Affiliated Zhongshan Hospital of Dalian University, Dalian 116 001, China 2 School of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang, China 3 The Nursing Department, Affiliated Zhongshan Hospital of Dalian University, Dalian, China *Corresponding author (Email, gkysgl@126.com)

Podophyllotoxin (PPT) and its derivatives exert significant anti-cancer activities, and one derivative etoposide is often utilized to treat various cancers in the clinic. The aim of the present study is to investigate the inhibitory effects of PPT on major cytochrome P450 (CYP) isoforms in human livers. Inhibition of CYP3A4, CYP2C9, CYP2C8, CYP2D6, CYP2E1 and CYP2A6 by PPT was investigated in the human liver microsomal system. Time-dependent inhibition of CYP3A4 by PPT was also evaluated. The results showed that PPT strongly exhibited inhibitory effects on CYP3A4 and CYP2C9 in a concentration-dependent manner. Half inhibition concentration (IC50) was 1.10.3 and 4.60.3 M for CYP3A4 and CYP2C9, respectively. Inhibition kinetic analysis showed that PPT exhibited competitive inhibition towards CYP3A4 and CYP2C9 with Ki of 1.6 and 2.0 M, respectively. Additionally, PPT exerted time-dependent inhibition towards CYP3A4 and the kinetic parameters were 4.42.1 M and 0.060.01 min1 for KI and kinact, respectively. Our experimental data indicate that potential drugdrug interaction (DDI) might exist when PPT is co-administered with the substrates which mainly undergo CYP3A4- or CYP2C9-mediated metabolism.
[Song J-H, Sun D-X, Chen B, Ji D-H, Pu J, Xu J, Tian F-D and Guo L 2011 Inhibition of CYP3A4 and CYP2C9 by podophyllotoxin: Implication for clinical drugdrug interactions. J. Biosci. 36 879885] DOI 10.1007/s12038-011-9143-9

1.

Introduction

Podophyllotoxin (PPT, figure 1) is the most abundant lignan extracted from podophyllin, which is a resin produced by species of the genera Podophyllum (Berberidaceae) (Imbert 1998). PPT has been used as medications by various cultures for over 1000 years (Slevin 1991). It has been used to treat viral condition, Condyloma acuminatum, which induces a variety of venereal warts (Sackett 1993). It is also employed as anti-malarial and anti-fungal agents with immune-modulating activities (Pugh et al. 2001). Another outstanding property of PPT is its anti-cancer activity. It could exert anti-tumour activity towards various types of tumors, including Kaposis sarcoma, malignant melanomas, non-Hodgkin and other lymphomas (Berkowitz et al. 2000), and lung cancer (Utsugi et al.
Keywords.

1996; Subrahmanyam et al. 1998). Additionally, the derivative of PPT etoposide also plays an important role for treatment of various cancer, including acute myeloid leukaemia, Hodgkins disease, lung cancer, gastric cancer, breast cancer and ovarian cancer (Radice et al. 1979; Johnson et al. 1991; Hande 1998). Drugdrug interaction (DDI) is one of the most important reasons for termination of development of promising new therapies, withdrawal of drugs from the market, or severe restrictions on the utilization of drugs (Wienkers and Heath 2005). Cytochrome P450 enzymes (CYPs) play an important role in the metabolic elimination of drugs and are implicated in a high proportion of DDIs (Lin and Lu 1998). Patients with cancers are at high risk for DDIs because anticancer drugs are often co-administered with other kinds of drugs, such as antiepileptic drugs and anticoagulants. For

Cytochrome P450 (CYP); drugdrug interaction (DDI); podophyllotoxin (PPT); time-dependent inhibition (TDI) J. Biosci. 36(5), December 2011, 879885, * Indian Academy of Sciences 879

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Table 1. Inhibition effects of PPT on six CYP isoforms in human liver microsomes CYP isoforms CYP2A6 CYP2C8 CYP2C9 CYP2D6 CYP2E1 CYP3A4 Probe reactions Coumarin 7-hydroxylation Paclitaxel 6a-hydroxylation Diclofenac 4-hydroxylation Dextronethorphan O-demethylation Chlorzoxazone 6-hydroxylation Testosterone 6-hydroxylation % control activity (remaining) 97.41.6 38.41.8 5.40.3 63.21.5 79.62.7 5.70.9 Positive control 8-methoxypsoralen Montelukast Sulfaphenazole Quinidine Clomethiazole Ketoconazole % control activity (remaining) 11.50.5 11.20.1 6.00.1 8.60.4 18.90.4 5.00.3

examples, anti-cancer drugs cyclophosphamide and ifosfamide are the inhibitors of CYP2B6. They can affect the elimination of bupropion, which is mainly metabolized by this CYP isoform. Therefore, when bupropion is co-dosed with cyclophosphamide or ifosfamide, monitoring of the patients response is required. Irinotecan exerts mixed inhibitory effects on CYP2C9 (Hanioka et al. 2002), which leads to a decrease in citalopram and results in an increased risk of rhabdomyolysis (Richards et al. 2003). The aim of the present study was firstly to investigate the inhibitory effects of PPT towards six major CYP isoforms. Furthermore, time-dependent inhibition of CYP3A4 and CYP2C9 was evaluated because this kind of inhibition could result in the inactivation of drug metabolizing enzymes and enzyme activities are only restored by de novo protein synthesis. All these results are important for investigation of CYPs inhibition and DDI associated with compounds containing core structure of PPT. 2. Materials and methods 2.1 Chemicals

2.2

Preparation and characterization of liver microsomes

The source of human liver microsomes (HLMs) has been previously reported (Zhang et al. 2006). Microsomes were prepared from liver tissue by differential ultracentrifugation (Zhang et al. 2006). Protein concentrations of the microsomal fractions were determined by the Lowry method using bovine serum albumin as a standard (Lowry et al. 1951). Determination of the content of CYPs was performed according to Omura and Sato (1964). 2.3 CYP probe substrate assays

Human liver microsomal coumarin 7-hydroxylation, paclitaxel 6-hydroxylation, diclofenac 4-hydroxylation, dextromethorphan O-demethylation, chlorzoxazone

PPT (purity98%) was purchased from Sichuan Weikeqi Bio-technology Co. Ltd (Sichuan, China). Paclitaxel, quinidine, D-glucose-6-phosphate, glucose-6-phosphate dehydrogenase, NADP + , sulfaphenazole, clomethiazole, furafylline, 8-methoxypsoralen and chlorzoxazone were obtained from Sigma-Aldrich (St Louis, MO, USA). 6hydroxypaclitaxel was purchased from BD Gentest Corp (Woburn, MA, USA). Ketoconazole (KTZ), coumarin, diclofenac, and dextromethorphan were purchased from ICN Biomedicals Inc (Aurora, Ohio, USA). Testosterone was purchased from Acros Organics (Geel, Belgium). Montelukast was purchased from Beijing Aleznova Pharmaceutical (Beijing, China). All other reagents were of high-performance liquid chromatography (HPLC) grade or the highest purity commercially available.
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Figure 1. The structure of PPT.

Inhibition of CYP isoforms by podophyllotoxin 6-hydroxylation and testosterone 6-hydroxylation activities were used as probe reactions for CYP2A6, CYP2C8, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, respectively. Briefly, the incubation system contained 100 mM potassium phosphate buffer (pH=7.4), NADPH-generating system (1 mM NADP+, 10 mM glucose-6-phosphate, 1 unit/ml of glucose-6-phosphate dehydrogenase, and 4 mM MgCl2), appropriate concentration of HLMs, respective probe substrate and PPT (or positive control inhibitor) in a final volume of 200 l. After 3 min pre-incubation at 37C, the reaction was initiated by adding NADPHgenerating system and terminated by adding 100 l acetonitrile (10% trichloroacetic acid for CYP2A6) with internal standard. The incubation conditions including substrate, protein concentration, incubation time, internal standard, and HPLC condition have been reported (Zhang et al. 2006). 2.4 Enzyme inhibition experiments

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To evaluate the inhibitory effect of PPT on six different human CYP isoforms, marker assays for each CYP isoform were performed in the presence of 100 M PPT. The concentrations of positive inhibitors used are as follows (Bjornsson et al. 2003): 2.5 M 8methoxypsoralen for CYP2A6, 1 M ketoconazole for CYP3A4, 10 M sulfaphenazole for CYP2C9, 10 M quinidine for CYP2D6, 50 M clomethiazole for CYP2E1, and 5 M montelukast for CYP2C8 (Walsky et al. 2005). For CYP isoforms that were strongly inhibited, half inhibition concentration (IC50) values were determined using various concentrations of PPT (20, 5, 2, 1, 0.5, 0.2, 0 M for CYP3A4 and 100, 80, 60, 40, 20, 10, 5, 1, 0 M for CYP2C9). The Ki value was obtained by incubating various probe substrates (50 M diclofenac and

Figure 2. Reversible inhibition of CYP3A4 by PPT. (A) Inhibition potential of PPT on testosterone 6-hydroxylation activity (CYP3A4). (B) Dixon plot of inhibition effect of PPT on testosterone 6-hydroxylation activity (CYP3A4). (C) Lineweaver-Burk plot of inhibition effect of PPT on testosterone 6-hydroxylation activity (CYP3A4). (D) Second plot of slopes from Lineweaver-Burk plot vs PPT concentrations. J. Biosci. 36(5), December 2011

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Jin-Hui Song et al. containing an NADPH-generating system and probe substrates. The concentrations of probe substrates were proximal to Km values. Further incubations were carried out to measure residual activity. 2.6 Estimation of inactivation constants

30100 M testosterone) and various concentrations of PPT (05 M for CYP3A4 and 020 M for CYP2C9). 2.5 Single point inactivation experiments

For CYP3A4 and CYP2C9, single point inactivation experiments were used to determine NADPH-dependent and preincubation-dependent inhibition as previously reported (Obach et al. 2007). In brief, pooled human liver microsomes (1 mg/ml) were incubated with PPT in the absence and presence of NADPH-generating system for 30 min at 37C. The concentration of PPT utilized was 10-fold as that which gave 25% inhibition under reversible inhibition situations. After incubation, an aliquot (20 l) was transferred to another incubation tube (final volume 200 l)

To determine the KI and kinact values for the inactivation of CYP3A4, six concentrations of PPT (0, 1, 2, 4, 10, 20 M) were incubated with pooled human liver microsomes (1 mg/ml) at 37C for 0 to 20 min. After pre-incubation, an aliquot (20 l) was transferred to another incubation tube (final volume 200 l) containing an NADPH-generating system and 200 M testosterone (for CYP3A4) to measure residual activity. Four times the Km concentrations of

Figure 3. Reversible inhibition of CYP2C9 by PPT. (A) Inhibition potential of PPT on diclofenac 4-hydroxylation activity (CYP2C9). (B) Dixon plot of inhibition effect of PPT on diclofenac 4-hydroxylation activity (CYP2C9). (C) Lineweaver-Burk plot of inhibition effect of PPT on diclofenac 4-hydroxylation activity (CYP2C9). (D) Second plot of slopes from Lineweaver-Burk plot vs PPT concentrations. J. Biosci. 36(5), December 2011

Inhibition of CYP isoforms by podophyllotoxin substrates were selected to minimize the reversible inhibition caused by PPT. To determine kobs (observed inactivation rate) values, the decrease in natural logarithm of the activity over time was plotted for each PPT concentration, and kobs values were described as the negative slopes of the line. Inactivation kinetic parameters were calculated using nonlinear regression of the data according to the equation 1 (Polasek and Miners 2007):

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did not exhibit time-dependent inhibition towards CYP2C9. Given the fact that time-dependent inhibition of CYP often produces more serious DDI than reversible inhibition, PPT-associated DDI mediated by inhibition of CYP3A4 would be stronger than DDI mediated by inhibition of CYP2C9. 4. Discussion

kobs

kinact I KI I
3. Results

Methylenedioxyphenyl compounds (benzodioxoles) have been demonstrated to exhibit inhibition towards CYP isoforms, including reversible inhibition and timedependent inhibition (Murray 2000). For example, the

3.1

In vitro inhibition of CYP isoforms by PPT

Inhibitory effects of PPT on six major CYP isoforms in HLMs were investigated using 100 M PPT. As shown in table 1, all positive control inhibitors showed strong inhibitory effects on the corresponding probe reactions with >80% of control activity inhibited. At 100 M PPT, the residual activities of CYP3A4, CYP2C9, CYP2A6, CYP2D6, CYP2C8 and CYP2E1 were by 5.70.9%, 5.4 0.3%, 97.4 1.6%, 63.21.5%, 38.4 1.8% and 79.6 2.7%, respectively. Inhibition kinetic study was carried out for CYP3A4 and CYP2C9, whose activities were strongly inhibited. As shown in figure 2, PPT inhibited testosterone 6-hydroxylation (CYP3A4) in a concentrationdependent manner with an IC50 of 1.10.3 M. LineweaverBurk and Dixon plots showed that the inhibition of CYP3A4 by PPT was best fit to a competitive way and KI value was calculated to be 1.6 M from second plot of the slopes from Lineweaver-Burk plots vs the concentrations of PPT. The results also demonstrated that PPT inhibited diclofenac 4-hydroxylation (CYP2C9) in a concentrationdependent manner with an IC 50 of 4.6 0.3 M. Lineweaver-Burk and Dixon plots suggested that PPT competitively inhibited CYP2C9 and KI was calculated to be 2.0 M. These results (figure 3) indicated that PPT might induce DDI with the compounds mainly undergoing CYP3A4, CYP2C9-mediated metabolism. 3.2 Time-dependent inhibition of CYP3A4 by PPT

Time- and NADPH-dependent inhibitions were also evaluated. When PPT was pre-incubated with HLM for 30 min in the presence of NADPH, the percentage of inhibition of CYP3A4 by PPT was increased by 24%. As calculated from the observed inactivation plots (figure 4), inactivation kinetic parameters (KI and kinact) for CYP3A4 were 4.4 2.1 M and 0.060.01 min1, respectively. However, PPT

Figure 4. Time-dependent inhibition of PPT toward CYP3A4. (A) Time- and concentration-dependent inhibition of CYP3A4 by PPT. (B) The hyperbolic plot of kobs of CYP3A4 vs PPT concentrations. J. Biosci. 36(5), December 2011

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selective serotonin reuptake inhibitor paroxetine contains a methylenedioxyphenyl group and exerts time-dependent inhibition towards CYP2D6 (Bertelsen et al. 2003). That leads to clinical DDIs with co-administered drugs whose clearances mainly depend on the activity of CYP2D6, such as desipramine (Brosen et al. 1993; Alderman et al. 1997), metoprolol (Hemeryck et al. 2000), risperidone (Spina et al. 2001) and atomoxetine (Belle et al. 2002). Deoxypodophyllotoxin (DPT) has a structure similar to that of PPT. Previous experiments demonstrated that DPT was metabolized by CYP3A4 and CYP2C19 in rat and HLMs and exhibited strongly competitive inhibition towards CYP3A4 and CYP2C9 (Lee et al. 2008, 2010). The experimental data obtained from our present study showed that PPT also exhibited competitive inhibition towards CYP3A4 and CYP2C9. These results are very helpful for deeper investigation of the relationship between structure and CYPs inhibitory capacity of compounds containing core structure of PPT. Additionally, like other methylenedioxyphenyl compounds, PPT also exhibits time-dependent inhibition towards CYP3A4, and kinetic parameters were 4.4 2.1 M and 0.06 0.01 min1 for KI and kinact, respectively. This kind of inhibition situation has not been found for DPT although its structure is very similar with PPT. Therefore, the detailed mechanism about how tiny changes of structures influence the time-dependent inhibition need to be investigated. All these prediction data indicated that co-administration of PPT might induce clinical DDI with substrates that mainly undergo CYP3A4, 2C9-mediated metabolism. Because the derivative of PPT etoposide is always combined with other anti-cancer drugs to treat various cancers, these data are also very valuable for implication for possible DDIs associated with etoposide. References
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MS received 17 June 2011; accepted 01 August 2011 ePublication: 23 September 2011

Corresponding editor: INDRANEEL MITTRA

J. Biosci. 36(5), December 2011