Inhibitory effects of calf thymus DNA on metabolism activity of CYP450 enzyme in human liver microsomes.

Drug Metab. Pharmacokinet. 29 (6): 475–481 (2014).

Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)

Regular Article Inhibitory Effects of Calf Thymus DNA on Metabolism Activity of CYP450 Enzyme in Human Liver Microsomes Shuoye YANG 1 , Zhixia Q IU 2, Qiuyang Z HANG 2, Jiayin C HEN 2 and Xijing C HEN 2, * 1 College of Bioengineering, Henan University of Technology, Zhengzhou, P. R. China Center of Drug Metabolism and Pharmacokinetics, College of Pharmacy, China Pharmaceutical University, Nanjing, P. R. China

2

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk Summary: The present study investigated the effect of calf thymus DNA (ctDNA) on human hepatic cytochrome P450s (CYP450s) in vitro. Specific substrate probes for each isoform, CYP1A2, 2C9, 2C19, 2D6 and 3A4, were incubated using pooled human liver microsomes with or without ctDNA, and liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) method was developed for the analysis of probe metabolites. Enzyme kinetics parameters K i and IC 50 values were estimated to determine the types and strength of inhibition. ctDNA could specifically inhibit the metabolism of CYP2C9 probe substrates, with the IC 50 = 0.9955 µg/ml, while it was not able to inhibit CYP1A2, CYP2C19, CYP2D6 or CYP3A4 (IC 50 > 100 µg/ml). The results showed that ctDNA was a potent inhibitor of CYP2C9 enzyme, and has the metabolic interaction potential with the model drugs which are metabolism substrates of CYP2C9. The inhibition mechanism study suggested ctDNA may inhibit CYP2C9 by decreasing the activity of CYP450 reductase. These findings indicated that when the medical agents catalyzed mainly by CYP2C9 were coadministered in vivo with adsorptive material in vitro, the potential inhibitory effect of ctDNA on enzyme activity and the following metabolism character changes of the former should be highly focused on. Keywords: calf thymus DNA; cytochrome P450; drug interactions; metabolism; inhibitory effect

reported to induce or inhibit CYP450 enzymes.5,6) Therefore, a great deal of effort is expended in new drug research, in order to avoid the DDI mediated by candidate compounds.7,8) For instance, to date, a certain number of new drugs have been withdrawn from the market because they potently inhibit CYP450, thus leading to adverse effects in patients who concurrently take a variety of other commonly prescribed medicines. Calf thymus DNA (ctDNA), as a biological macromolecule compound, is one of the absorptive materials which cleans up leprosy bacillus through effective physical adsorption; thus it is used to purify patients’ blood in vitro and treat leprosy by hemodialysis.9,10) We investigated the in vivo pharmacokinetics and disposition property of ctDNA in a previous study, to evaluate its safety of use as an immunoadsorptive carrier material for therapy.11,12) Although it was proved ctDNA can be effectively eliminated in plasma and would not accumulate in the body of animal, in the practical application, the patients who have leprosy or systemic lupus erythematosus (SLE) may undergo synergistic cure with other drugs together with ctDNA, or other kind of drugs

Introduction Nowadays, drug-drug interactions (DDI) are of great interest to scientists involved in drug development, clinical research and toxicology evaluation, and already becoming an important branch of pharmacology and pharmacokinetics. The majority of these interactions involve cytochrome P450 (CYP450). CYP450 enzymes consist of the superfamily of hemeproteins which catalyze the oxidative metabolism of substrates. For the broad range of substrate specificity, CYP enzymes play an important role in Phase I metabolism involved in the biotransformation of drugs and other xenobiotics, as well as some endogenous substrates.1,2) Among numerous CYP enzymes identified to date, five human liver CYP isoforms, CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, have been confirmed to be the most relevant isozymes in the metabolism of clinically commonly used drugs.3,4) In most cases, inhibition of CYP450 activities is the most common mechanism resulting in DDI, and may lead to potential toxicity or alter the efficacy of drugs. Some herbs and natural remedies have been

Received November 29, 2013; Accepted June 30, 2014 J-STAGE Advance Published Date: July 15, 2014, doi:10.2133/dmpk.DMPK-13-RG-131 *To whom correspondence should be addressed: Xijing CHEN, Ph.D., Center of Drug Metabolism and Pharmacokinetics, College of Pharmacy, China Pharmaceutical University, 24# Tong Jia Xiang Street, Nanjing 210009, P. R. China. Tel. ©86-25-83271286, Fax. ©86-25-83271335, E-mail: [email protected] This work was partially supported by the Jiangsu Provincial Promotion Foundation for the Key Lab Drug Metabolism and Pharmacokinetics of China (No: BM2012012). 475

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were simultaneously or successively used against other diseases and medical conditions. Therefore, DDI may occur if some ctDNA falls off the immunoadsorption column and goes into the blood circulatory system. To ensure safe application, it is essential to conduct comprehensive inhibitory analysis for ctDNA with major drug-metabolizing CYPs. The present study was carried out to determine the potential of a nucleic acid compound, ctDNA, to inhibit the activity of human CYP enzymes that are most commonly associated with the metabolic elimination of drugs: CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, followed by a full-scale characterization of the kinetics and type of inhibition using classical probe substrates. Materials and Methods Chemicals and reagents: The ctDNA was provided by Jianfan Biotechnology Company (Zhuhai, China). Phenacetin, diclofenac, mephenytoin, dextromethorphan, midazolam and testosterone were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) and used as probe substrates. Acetaminophen, 4-hydroxydiclofenac, 4-hydroxymephenytoin, dextrorphan, 1-hydroxymidazolam and 6¢-hydroxytestosterone were purchased from Sigma-Aldrich (St. Louis, MO) and used as metabolites of specific substrates. Chlorzoxazone, caffeine, nimodipine and diazepam were purchased from National Institute for the Control of Pharmaceutical and Biological Products and used as internal standards (ISs). The ¡-naphthoflavone, sulphaphenazole and quinidine were purchased from Sigma-Aldrich, and ticlopidine and ketoconazole were from National Institute for the Control of Pharmaceutical and Biological Products, all of which were used as inhibitors. NADPH was purchased from Sigma-Aldrich. All other reagents were of analytical grade. Human liver microsomes: Human liver microsomes (HLMs) were provided by the Research Institute for Liver Disease Co. (RILD) (Shanghai, China). The microsomes were prepared from ten individual human donor livers. Enzyme kinetics essay: All the standard solutions of each substrate were manipulated by serial dilution to concentration gradients: phenacetin, 20–4,000 µmol/L; diclofenac, 50–4,000 µmol/L; mephenytoin, 50–2000 µmol/L; dextromethorphan, 1– 500 µmol/L; midazolam, 1–400 µmol/L; testosterone, 50–4,000 µmol/L. After incubation with HLMs at 37°C for optimizing time, the formation rates for specific metabolites were observed and analyzed to estimate the kinetic parameters of substrates. Optimizing of incubation time: The final concentrations of microsome proteins and substrates in reaction systems were fixed; metabolism reactions were initiated and terminated at different times to determine the formation amounts of individual metabolites, to evaluate the linear timelines of metabolism mediated by CYPs for each probe substrate. Inhibition of CYP450s by ctDNA: The CYP enzymatic activities were determined by O-deethylation of phenacetin for CYP1A2, 4-hydroxylation of diclofenac for CYP2C9, 4-hydroxylation of mephenytoin for CYP2C19, O-demethylation of dextromethorphan for CYP2D6, 1-hydroxylation of midazolam and 6¢-hydroxylation of testosterone for CYP3A4. For each inhibition study, preliminary incubation experiments were carried out using a single CYP-specific substrate concentration lower than its Km (the Michaelis constant), specifically, phenacetin-20 µM; diclofenac-10 µM; mephenytoin-20 µM; dextromethorphan-5 µM;

midazolam-2 µM and testosterone-20 µM, in the presence of a range of ctDNA concentrations (1.0–200 µg/ml) in the incubation mixture. Probe substrates were incubated with HLMs (0.2 mg/ml), 5 mM MgCl2 and 1.0 mM NADPH in a total volume of 200 µl of 150 mM phosphate buffer (pH 7.4), in the presence and absence of ctDNA. After being preincubated for 5 min in an incubator shaker at 37°C, the reactions were initiated by adding NADPH. Known specific inhibitors were used in parallel as positive controls: ¡-naphthoflavone for CYP1A2, sulfaphenazole for CYP2C9, ticlopidine for CYP2C19, quinidine for CYP2D6 and ketoconazole for CYP3A4. The Ki (equilibrium dissociation constant of reversible inhibitor) values of ctDNA were not determined if their IC50 (the half maximal inhibitory concentration of a substance) was higher than 100 µM. Inhibitor and substrate concentrations for determining Ki value were selected based on the preliminary study. The range of concentrations of phenacetin (0–0.6 µM) and ctDNA (1.0–200 µg/ml) were used for estimation of Ki value. All measurements were performed in triplicate. Analytical methods: Acetaminophen After vortexing and centrifuging at 12,000 rpm for 5 min, the supernatant was taken and evaporated at 40°C to dryness, and then the samples were reconstituted by adding 100 µl of 25 ng/ml caffeine dissolved in methanol and a 20 µl aliquot was injected for analysis by liquid chromatography and tandem mass spectrometry (LC-MS/MS). The chromatographic separation was performed using a Hypersil ODS2 column (150 mm©2.0 mm, 5 µm, VP-ODS, Shimadzu, Tokyo, Japan). The mobile phase comprising methanol0.2% methanoic acid solution at a flow rate of 0.35 ml/min and gradient elution was applied. The analyte was monitored by selected ion monitoring of m/z 109.9 (acetaminophen) and m/z 138.0 (IS) with the tandem mass spectrometer operated in the positive ion mode using a TSQ Quantum Access triple quadrupole mass spectrometer. Acetaminophen was quantitated with a linear range from 0.005 to 2 µg/ml. 4-Hydroxydiclofenac After being mixed with 20 µl of 2.0 ng/ml chlorzoxazone, the samples were shaken and centrifugated at 12,000 rpm for 5 min, and the supernatant was taken and was evaporated to dryness. Then the samples were redissolved by adding 200 µl of the mobile phase, which comprised methanol-water (70:30, v/v) and a 5 µl aliquot was injected for analysis by LC-MS/MS. The chromatographic separation was performed using a BDS HYPERSIL C18 column (50 mm©2.1 mm, 2.4 µm, Thermo Scientific, Waltham, MA) at a flow rate of 0.3 ml/min. The analyte was monitored by selected ion monitoring of m/z 270.1 (4-hydroxydiclofenac) and m/z 132.1 (IS) with the tandem mass spectrometer operated in the negative ion mode. 4-Hydroxydiclofenac was quantitated with a linear range from 0.02 to 2 µg/ml. 4-Hydroxymephenytoin The reaction was stopped by adding 20 µl of 0.05 µg/ml diazepam. The samples were shaken and centrifuged at 12,000 rpm for 5 min, the supernatant was taken and the organic phase was evaporated to dryness. Then the samples were redissolved by adding 200 µl of the mobile phase, which comprised methanolwater (all contained 0.1% methanoic acid) and a 10 µl aliquot was injected for analysis by LC-MS/MS. Chromatography was done using a BDS HYPERSIL C18 column (50 mm©2.1 mm, 2.4 µm, Thermo Scientific) at a flow rate of 0.5 ml/min with gradient elution applied. The analyte was monitored by selected ion moni-


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toring of m/z 150.1 (4-Hydroxymephenytoin) and m/z 193.2 (IS) with the tandem mass spectrometer operated in the positive ion mode. 4-Hydroxymephenytoin was quantitated with a linear range from 0.001 to 0.1 µg/ml. Dextrorphan After vortexing and centrifuging at 12,000 rpm for 5 min, the supernatant was taken and evaporated. The residue was reconstituted by adding 100 µl of 2 ng/ml nimodipine dissolved in methanol and a 10 µl aliquot was injected for analysis by LC-MS/ MS. Chromatography was done using a Hypersil ODS2 column (150 mm©2.0 mm, 5 µm, VP-ODS, Shimadzu). The mobile phase comprising methanol-0.1% ammonium acetate solution at a flow rate of 0.35 ml/min and gradient elution was applied. The analyte was monitored by selected ion monitoring of m/z 132.9 (dextrorphan) and m/z 343 (IS) with the tandem mass spectrometer operated in the positive ion mode. Dextrorphan was quantitated with a linear range from 0.001 to 0.5 µg/ml. 1-Hydroxymidazolam The reaction was stopped by adding 200 µl of 0.05 µg/ml diazepam. The samples were shaken and centrifuged at 12,000 rpm for 10 min, and 5 µl of supernatant was taken and injected for analysis by LC-MS/MS. The chromatographic separation was performed using a Xtimate C18 column (100 mm©2.1 mm, 5 µm; Welch Material, Inc., Ellicott, MD) and equilibrated in a gradient mobile phase of water and acetonitrile at a flow rate of 0.3 ml/min. The analyte was monitored by selected ion monitoring of m/z 168.4 (1-hydroxymidazolam) and m/z 193.2 (IS) with the tandem mass spectrometer operated in the positive ion mode. 1-Hydroxymidazolam was quantitated with a linear range from 0.002 to 0.5 µg/ml. 6¢-Hydroxytestosterone After vortexing and centrifuging at 12,000 rpm for 3 min, the supernatant was taken and evaporated to dryness, and then the samples were redissolved by adding 100 µl of 25 ng/ml diazepam. A 10 µl aliquot was injected for analysis by LC-MS/MS and a BDS HYPERSIL C18 column (50 mm©2.1 mm, 2.4 µm, Thermo Scientific) was used for chromatographic separation. The gradient mobile phase comprising methanol-water (all contained 0.1% methanoic acid) was applied with the flow rate of 0.3 ml/min. The analyte was monitored by selected ion monitoring of m/z 269.2 (6¢-hydroxytestosterone) and m/z 193.2 (IS) with the tandem mass spectrometer operated in the positive ion mode. 6¢-Hydroxytestosterone was quantitated with a linear range from 0.01 to 0.5 µg/ml. Inhibition mechanism study: NADPH-CYP450 reductase (POR) is the only flavoprotein that mediates electron transfer from NADPH to CYP enzymes. The inhibition for POR activity will result in disruption of electron transfer, and thus, result in a profound decrease of hepatic drug metabolism. POR activity assay was performed by reduction of cytochrome C in NADPH. The reaction was performed in 0.025 mg/ml protein, 0.3 mmol/L NADPH, 0.9 mg/ml cytochrome C and 300 mmol/L K3PO4(PH7.4). Two, 10 and 50 µg/ml ctDNA were co-incubated and sampling took place at 0.5, 1 and 2 h, respectively. The spectra and peak scans were carried out at 550 nm within 3 min; the activity of POR in protein was calculated according to the value ¾ = 27.8 mmol/L/cm. Data analysis: The Ki values obtained from secondary Lineweaver–Burk plots were used as initial estimates for the determination of the exact Ki values by nonlinear least square regression analysis using GraphPad (Prism5.0). The data were all statistically processed by one-way analysis of variance.

Results Method validation: The LC-MS/MS method described above showed good separation of analyte and internal standard from other indigenous substance in HLMs; thus the developed method had high specificity with no interference for sample determination. The calibration curves were linear for all the metabolites in the selected concentration range with the correlation coefficients (r) higher than 0.99. The intra-day and inter-day precisions were below 15%, and the accuracies were within 85–115%. The results were all within the acceptable range and in accordance with the requirements for biological sample determination. Optimizing of incubation time for probe substrates: The optimizing results for incubation time investigated were 20, 15, 30, 15, 5, and 10 min for phenacetin, diclofenac, mephenytoin, dextromethorphan, midazolam and testosterone, respectively, indicating each special compound selected as probe substrate had a different optimal incubation time. Enzyme kinetics study: The corresponding initial velocities of enzyme reaction for each concentration of substrate were calculated. In the range of concentrations used in this study, the reaction kinetics from six probe substrates characterized in HLMs all accorded with the dynamic characteristics of Michaelis-Menten hyperbola, which are shown in Figure 1. The kinetics parameters, Km and Vmax, were obtained by nonlinear regression and are listed in Table 1. Inhibition effect of specific inhibitors on CYP450s: The six probe substrates were co-incubated with their own specific inhibitor in HLMs. The formation rates of metabolites at a variety of substrate and inhibitor concentrations were determined and Dixon plots for the inhibition of CYP isoforms were constructed, as presented in Figure 2. The inhibition rate constant Ki of each inhibitor for CYP isoforms was determined based on the analysis of nonlinear regression and GraphPad software for inhibition data, and is presented in Table 2. The results showed the Ki values for specific substrates of major CYP isoforms obtained in this study were in general agreement with the values in previously reported literature. Therefore, the in vitro test model can be well used to evaluate the effect of ctDNA on the activity of human CYP enzymes. Inhibition of CYP450 activities by ctDNA in HLMs: The capabilities of ctDNA to inhibit individual CYP enzymes were examined by the probe reaction assays with HLMs. IC50 values were determined by plotting with the formation rates of each probe metabolite in the incubation system versus the logarithm of the concentration gradient of ctDNA, and are listed in Table 3. It could be inferred that ctDNA strongly inhibited the reaction catalyzed by CYP2C9 in HLMs, with the IC50 value of 0.9955 µg/ml. For the remaining four isoforms, CYP1A2, CYP2C19, CYP2D6 and CYP3A4, the obtained IC50 values of ctDNA were all higher than 100 µg/ml, indicating their catalytic activity in vivo would not be inhibited by ctDNA. This suggests that, if ctDNA falls off and enters the circulatory system, it may interact with the drugs which are metabolized by CYP2C9 residing in bodies of patients. Furthermore, we conducted an inhibition experiment using smaller nucleic acid compounds from ctDNA to clarify the molecules that in fact inhibit CYP2C9. The different batches of ctDNA were dissolved and laid at 50°C for 0.5, 1, 2 and 4 h, with the concentrations of 5 and 20 µg/ml. Samples were used to evaluate the inhibitory activity. The results showed that the small molecules


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Fig. 1. Catalyzed dynamic characteristics of CYP450-specific substrates in HLMs A: phenacetin, B: diclofenac, C: mephenytoin, D: dextromethorphan, E: midazolam, F: testosterone.

Table 1. Kinetic parameters of CYP450-specific substrates in HLMs CYP450s

Substrates

CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4 CYP3A4

phenacetin diclofenac mephenytoin dextromethorphan midazolam testosterone

Km (µmol/L) 94.06 9.801 27.63 5.901 8.788 52.76

Vmax (pmol/min/mg protein) 185.9 908.8 13.25 1,165 616.7 1,378

derived from ctDNA had no strong inhibitory effects on CYP2C9, since all the IC50 values were higher than 100 µg/ml and there was no significant difference (p > 0.05) between nucleic acid molecules resulting from multiple batches of ctDNA at different incubation times. Inhibition mechanism study: The co-incubation with ctDNA showed a significant decrease in POR activity, which dropped with the time; as for the 2 µg/ml ctDNA group, it decreased POR activity by about 26%. The corresponding rate was up to approximately 60% while ctDNA concentration was 50 µg/ml (Fig. 3). Thus it can be inferred that POR activity was inhibited by ctDNA in a concentration- and time-dependent manner. This result suggested that the electron transfer activity can be inhibited by ctDNA significantly, and further, lead to the decreased metabolism activity of enzymes.

Discussion When medical products are administered concurrently, hepatic and intestinal drug metabolizing enzymes such as CYP450 maybe be induced or inhibited, which is regarded as the major mechanism for the enhanced or reduced bioavailability of drugs.13,14) Owing to altered residue time and clearance, the pharmaceutical potency of one substance will be affected by another, leading to an exaggeration of toxicity or a diminished efficacy and the resulting totally different clinical effectiveness.15,16) CYP2C9 is one of the major CYP450 enzymes that is associated with the metabolism of a wide range of therapeutic compounds, such as prostanoids, steroid hormones, anticoagulants and nonsteroidal anti-inflammatory drugs, especially those with a narrow therapeutic index.17–20) Until recently, although a large number of studies concerning the effect of chemicals and herbal medicine on CYP enzymes has been performed, the information available in the literature about DDIs arising from biotechnology medicines has been extremely scarce. In this study, LC-MS/MS determination methods for probe metabolites of CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 were developed. The methods were fully validated to meet the requirements of analysis in biological samples, and can be well applied to study inhibition toward CYP enzymes activities. On these bases, we examined the potential inhibition for the metabolism-based drug interaction arising from ctDNA. Each specific probe substrate of CYPs was incubated in HLM systems with ctDNA to evaluate its inhibition by measuring the activities of


Inhibition of ctDNA on Metabolism Activity of CYP450 Enzyme

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Fig. 2. Dixon plots illustrating the effects of a specific inhibitor on the metabolism of each probe substrate in human liver microsomes, by using the formation rates of metabolites versus the inhibitor concentrations Inhibitors: A: ¡-naphthoflavone, B: sulfaphenazole, C: ticlopidine, D: quinidine, E and F: ketoconazole; metabolizing reactions: A: phenacetin to acetaminophen, B: diclofenac to 4-hydroxydiclofenac, C: mephenytoin to 4-hydroxymephenytoin, D: dextromethorphan to dextrorphan, E: midazolam to 1-hydroxymidazolam, F: testosterone to 6¢-hydroxytestosterone. Each point represents the mean of triplicate determinations.

Table 2. The Ki values of the suppression effect for CYP450-specific substrates CYP450s

Substrates

Inhibitors

CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4 CYP3A4

phenacetin diclofenac mephenytoin dextromethorphan midazolam testosterone

¡-naphthoflavone sulphaphenazole ticlopidine quinidine ketoconazole ketoconazole

Ki(µmol/L) Measured Reference values valuea 0.0068 0.01 0.31 0.3 1.17 1.2 0.12 0.027–0.4 0.102 0.0037–0.18 0.096

a

The reference values are all drawn from the official website of the Food and Drug Administration: http://www.fda.gov/cder/drug/drug interactions/default. htm

Fig. 3. The reductive activity of POR estimated by NADPH-dependent cytochrome C reduction The activity was calculated and expressed in the presence of different concentrations of ctDNA (n = 3).

Table 3. The IC50 values for the inhibition of calf thymus DNA on CYP450 activities Substrates phenacetin diclofenac mephenytoin dextromethorphan midazolam testosterone

CYP450 isoforms CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4 CYP3A4

IC50 (µg/ml) >100 0.9955 >100 >100 >100 >100

marker reactions. The Ki and IC50 values of ctDNA toward CYP450 isoforms were determined. The ctDNA showed a strong competitive type of inhibitory effect on CYP2C9-catalyzed diclofenac 4hydroxylation, whereas it was not able to inhibit other CYP450 isoforms. Once ctDNA gets into a body, it may cause drug interaction in humans when co-administered with drugs that are metabolized primarily by CYP2C9, suggesting care must be taken concerning its potential inhibition and changes to the metabolism character of therapeutic agents catalyzed by CYP2C9.21) Moreover,


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since ctDNA is a mixture composed of large amounts of nucleic acid molecules without identical molecular weight, the mass concentration (µg/ml) was used to calculate IC50, substituting the molar concentration (µM) conventionally applied to study in vitro inhibition.22) As a known highly active inductor for CYP2C-subtype enzymes, rifampicin is also an effective and widely used drug for treating leprosy.23,24) If rifampicin and ctDNA are used together in vivo and in vitro to treat against leprosy, the metabolizing activity of CYP2C9 maybe be positively and negatively regulated simultaneously once ctDNA has fallen into a patient’s body; thus these effects from exogenous substances on CYP2C9 will be complicated accordingly. Cortisone, one of the glucocorticoids, which has a powerful anti-inflammatory effect and is commonly used in adjuvant medical therapy, can be well used to cure various inflammatory responses, allergic urticaria, lupus erythematosus and leprosy.25,26) It was reported that combining it with inductors of metabolism enzymes would result in accelerated catabolizing of adrenal cortex hormone, suggesting cortisone is also catalyzed and metabolized by CYP2C-subtypes. Therefore, when used in combination with virus adsorbent ctDNA, care must be taken concerning the possible result that cortisone is catabolized more slowly because of the weakened enzyme activity. Further, in practical application, after hemodialysis with absorptive columnloaded carrier materials, purified blood is returned to the body. Sometimes anticoagulants are applied to promote smoother blood stream back into the circulatory system for better blood circulation. Warfarin is a typical frequently employed coumarin agent, and is also a special substrate of CYP2C9.13,16) Hence, the possible DDI with ctDNA is a problem that cannot be ignored if used for anticoagulation. In theory, severe enzyme inhibition occurs when the inhibitor concentration ([I]) present at the active site achieves or surpasses the Ki. Whether an in vivo interaction appears mainly depends on the [I]/Ki ratio in which [I] represents the mean Cmax value at the steady-state level for the proposed total drug dose (including bound and unbound forms). The likelihood of DDI increases as the ratio increases. Although quantitative predictions for in vivo interactions based on in vitro studies only are not completely effective or reliable, the significance of the metabolic interaction of ctDNA should be confirmed and may help to prioritize the in vivo interaction evaluations. It is generally accepted that the substrates and inhibitors of CYP2C9 are mostly small molecules due to the smaller cavity in their active sites supported by the crystal analysis for CYP2C9 enzymes. Therefore, a macromolecule such as ctDNA may not be an actual or only substrate of CYP2C9 because crossing hepatocyte membranes makes it hard for it to bind to the active sites. Meanwhile, a large part of ctDNA will be digested to nucleotides or nucleic acids in the gastrointestinal tract and absorbed. Not only ctDNA but the nucleic acids will be exposed to CYP450s. The detailed study of inhibition towards CYP2C9 was performed based on ctDNA being degraded to relatively smaller molecule nucleic acids at high temperature. That the concomitant as well as separated small molecule in ctDNA has no inhibition potential for CYP2C9 enzyme was suggested by the results. As the electron origin for CYP enzyme, POR plays an important role in the oxidative metabolism process of CYPs.27) Cytochrome C is the most frequently used electron receptor in determining POR activity, although it may not be the endogenous substrate of

POR.28) The results showed that the co-incubation with ctDNA would result in decreased activity of POR concentration-dependently to some extent, which further interferes in the electron transfer process from POR to CYP2C9. The other CYP enzyme isoforms may not be affected by this inhibition arising from ctDNA. Therefore, it could be inferred cautiously that CYP2C9 was inhibited not by binding to its active sites, but by causing interference with the interaction between 2C9 enzymes and CYP450 reductase. In summary, systematic research was conducted in this study to evaluate and predict drug interactions promoted by ctDNA. Our data indicated that ctDNA potentially possessed high potency to inhibit the activity of CYP enzymes. Careful monitoring would be indispensable for the concomitant use of ctDNA with other drugs that are metabolized primarily by CYP2C9. Coupled with our earlier pharmacokinetics study in animals, we have provided the comprehensive in vivo and in vitro pharmacokinetics information for ctDNA, which can be used as beneficial guidance for the safe and reasonable application of this absorptive material in therapeutic practice. References 1) 2) 3) 4) 5)

6) 7)

8) 9) 10) 11)

12)

13)

14)

Wrighton, S. A., Van den Branden, M. and Ring, B. J.: The human drug metabolizing cytochromes P450. J. Pharmacokinet. Biopharm., 24: 461– 473 (1996). Xu, X., Liu, H. Y., Liu, L., Xie, L. and Liu, X. D.: The influence of a newly developed quinolone: antofloxacin, on CYP activity in rats. Eur. J. Drug Metab. Pharmacokinet., 33: 1–7 (2008). Wienkers, L. C. and Heath, T. G.: Predicting in vivo drug interactions from in vitro drug discovery data. Nat. Rev. Drug Discov., 4: 825–833 (2005). Hodgson, E. and Rose, R. L.: The importance of cytochrome P450 2B6 in the human metabolism of environmental chemicals. Pharmacol. Ther., 113: 420–428 (2007). Madgula, V. L., Ali, Z., Smillie, T., Khan, I. A., Walker, L. A. and Khan, S. I.: Alkaloids and saponins as cytochrome p450 inhibitors from blue cohosh (Caulophyllum thalictroides) in an in vitro assay. Planta Med., 75: 329–332 (2009). Usia, T., Iwata, H., Hiratsuka, A., Watabe, T., Kadota, S. and Tezuka, Y.: CYP3A4 and CYP2D6 inhibitory activities of Indonesian medicinal plants. Phytomedicine, 13: 67–73 (2006). Kalgutkar, A. S., Obach, R. S. and Maurer, T. S.: Mechanism-based inactivation of cytochrome P450 enzymes: chemical mechanisms, structure–activity relationships and relationship to clinical drug–drug interactions and idiosyncratic adverse drug reactions. Curr. Drug Metab., 8: 407–447 (2007). Pelkonen, O., Turpeinen, M., Hakkola, J., Honkakoski, P., Hukkanen, J. and Raunio, H.: Inhibition and induction of human cytochrome P450 enzymes: current status. Arch. Toxicol., 82: 667–715 (2008). Belak, M., Borberg, H., Jimenez, C. and Oette, K.: Technical and clinical experience with protein A immunoadsorption columns. Transfus. Sci., 15: 419–422 (1994). Braun, N. and Bosch, T.: Immunoadsorption, currents status and future developments. Expert Opin. Investig. Drugs, 9: 2017–2038 (2000). Yang, S. Y., Zhang, Q. Y., Chen, J. Y., Han, D. E., Zhao, D. and Chen, X. J.: Pharmacokinetics and disposition study of calf thymus DNA in rats by applying 3H-labeling method. J. Pharm. Biomed. Anal., 64–65: 35–39 (2012). Yang, S. Y., Amer, T., Wang, X., Song, H. L. and Chen, X. J.: Pharmacokinetics study of calf thymus DNA in rats and beagle dogs with 3 H-labeling and tracing method. J. Pharm. Biomed. Anal., 88: 60–65 (2014). Muszkat, M., Blotnik, S., Elami, A., Krasilnikov, I. and Caraco, Y.: Warfarin metabolism and anticoagulant effect: A prospective, observational study of the impact of CYP2C9 genetic polymorphism in the presence of drug-disease and drug-drug interactions. Clin. Ther., 29: 427– 437 (2007). Zhou, Q., Matsumoto, S., Ding, L. R., Fischer, N. E. and Inaba, T.: The comparative interaction of human and bovine serum albumins with CYP2C9 in human liver microsomes. Life Sci., 75: 2145–2155 (2004).


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16) 17)

18) 19)

20) 21)

Ikeda, T., Aoyama, S., Tozuka, Z., Nozawa, K., Hamabe, Y., Matsui, T., Kainuma, M., Hasegawa, S., Maeda, K., et al.: Microdose pharmacogenetic study of 14C-tolbutamide in healthy subjects with accelerator mass spectrometry to examine the effects of CYP2C9/3 on its pharmacokinetics and metabolism. Eur. J. Pharm. Sci., 49: 642–648 (2013). Yong, W. P., Kim, T. W., Undevia, S. D., Innocenti, F. and Ratain, M. J.: R(+)XK469 inhibits hydroxylation of S-warfarin by CYP2C9. Eur. J. Cancer, 45: 1904–1908 (2009). Zhang, Z. Y., Kerr, J., Wexler, R. S., Li, H. Y., Robinson, A. J., Harlow, P. P. and Kaminsky, L. S.: Warfarin analog inhibition of human CYP2C9-catalyzed S-warfarin 7-hydroxylation. Thromb. Res., 88: 389– 398 (1997). Al-Jenoobi, F. I.: Effects of some commonly used Saudi folk herbal medications on the metabolic activity of CYP2C9 in human liver microsomes. Saudi Pharm. J., 18: 167–171 (2010). Danielson, M. L., Desai, P. V., Mohutsky, M. A., Wrighton, S. A. and Lill, M. A.: Potentially increasing the metabolic stability of drug candidates via computational site of metabolism prediction by CYP2C9: The utility of incorporating protein flexibility via an ensemble of structures. Eur. J. Med. Chem., 46: 3953–3963 (2011). Banu, H., Renuka, N. and Vasanthakumar, G.: Reduced catalytic activity of human CYP2C9 natural alleles for gliclazide: molecular dynamics simulation and docking studies. Biochimie, 93: 1028–1036 (2011). Wang, X., Cheung, C. M., Lee, W. Y., Or, P. M. and Yeung, J. H.: Major tanshinones of Danshen (Salvia miltiorrhiza) exhibit different modes of inhibition on human CYP1A2, CYP2C9, CYP2E1 and CYP3A4

22)

23) 24) 25) 26) 27)

28)

481

activities in vitro. Phytomedicine, 17: 868–875 (2010). Or, P. M., Lam, F. F., Kwan, Y. W., Cho, C. H., Lau, C. P., Yu, H., Lin, G., Lau, C. B., Fung, K. P., et al.: Effects of Radix Astragali and Radix Rehmanniae, the components of an anti-diabetic foot ulcer herbal formula, on metabolism of model CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 probe substrates in pooled human liver microsomes and specific CYP isoforms. Phytomedicine, 19: 535–544 (2012). Gangadhar, K. N., Changsan, V., Buatong, W. and Srichana, T.: Phase behavior of rifampicin in cholesterol-based liquid crystals and polyethylene glycol. Eur. J. Pharm. Sci., 47: 804–812 (2012). Abed, M., Towhid, S. T., Shaik, N. and Lang, F.: Stimulation of suicidal death of erythrocytes by rifampicin. Toxicology, 302: 123–128 (2012). Shi, L., Maser-Gluth, C. and Remer, T.: Daily urinary free cortisol and cortisone excretion is associated with urine volume in healthy children. Steroids, 73: 1446–1451 (2008). Nomura, S., Fujitaka, M., Sakura, N. and Ueda, K.: Circadian rhythms in plasma cortisone and cortisol and the cortisone/cortisol ratio. Clin. Chim. Acta, 266: 83–91 (1997). Wang, S. L., Han, J. F., He, X. Y., Wang, X. R. and Hong, J. Y.: Genetic variation of human eytochrome P450 reductase as a potential biomarker for mitomycin C-induced cytotoxicity. Drug Metab. Dispos., 35: 176– 179 (2007). Pandey, A. V., Flück, C. E. and Mullis, P. E.: Altered heme catabolism by heme oxygenase-1 caused by mutations in human NADPH cytochrome P450 reductase. Biochem. Biophys. Res. Commun., 24: 374–378 (2010).


Inhibitory effect of six herbal extracts on CYP2C8 enzyme activity in human liver microsomes.

CYP450 Enzyme-Mediated Metabolism of TCAS and Its Inhibitory and Induced Effects on Metabolized Enzymes in Vitro.

Inhibitory Effects of Dimethyllirioresinol, Epimagnolin A, Eudesmin, Fargesin, and Magnolin on Cytochrome P450 Enzyme Activities in Human Liver Microsomes.

Inhibitory effects of cedrol, β-cedrene, and thujopsene on cytochrome P450 enzyme activities in human liver microsomes.

Effects of tanshinones from Salvia miltiorrhiza on CYP2C19 activity in human liver microsomes: enzyme kinetic and molecular docking studies.

Metabolism and Metabolic Inhibition of Xanthotoxol in Human Liver Microsomes.

In Vitro Metabolism of Flucetosulfuron by Human Liver Microsomes.

Further studies on partially purified calf thymus DNA polymerase a.

DNA substrate specificity of DNA helicase E from calf thymus.

Inhibitors of imipramine metabolism by human liver microsomes.

Metabolism of diazepam and related benzodiazepines by human liver microsomes.

Metabolism of ketamine stereoisomers by human liver microsomes.

Cloning of calf thymus satellite I DNA in Escherichia coli.

The metabolism of tramadol by human liver microsomes.

Metabolism of coumarin by rat, gerbil and human liver microsomes.

Structural transitions of calf thymus DNA in concentrated LiCl solutions.

Resolution of synthetic Holliday junctions in DNA by an endonuclease activity from calf thymus.

Characterization of paracetamol UDP-glucuronosyltransferase activity in human liver microsomes.

Inhibitory effects of kiwifruit extract on human platelet aggregation and plasma angiotensin-converting enzyme activity.

Study on interactions of aminoglycoside antibiotics with calf thymus DNA and determination of calf thymus DNA via the resonance Rayleigh scattering technique.

Relationship between activities of cytochrome P-450 monooxygenases in human placental microsomes and binding of benzo(a)pyrene metabolites to calf thymus DNA.

Probing the binding mode of psoralen to calf thymus DNA.

In vitro effect of fluoroquinolones on theophylline metabolism in human liver microsomes.

Subunit SA3 is not mandatory for the activity of calf thymus DNA-dependent RNA polymerase AI.