http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2014; 52(6): 681–687 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2013.864685
ORIGINAL ARTICLE
Effects of Alismatis rhizome on rat cytochrome P450 enzymes Yi Huang1, Shuang-li Zheng2, Zhi-sheng Xu3, and Yao Hou4
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1
Department of General Surgery of Wenzhou People’s Hospital, Wenzhou, China, 2Department of Pediatric Surgery of the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, China, 3Department of Hematology of the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, China, and 4Department of Ultrasound Imaging of the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China
Abstract
Keywords
Context: Alismatis rhizome (RA) (Water Plantain Family, also called ‘‘Zexie’’ in Chinese), one of the commonly used components of traditional Chinese medicines, is derived from the dried rhizomes of Alisma orientalis (Sam.) Juzep. (Alismataceae). Objective: This study explores the RA influences on rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9, CYP2E1 and CYP3A4) by using cocktail probe drugs in vivo. Materials and methods: A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (20 mg/kg), tolbutamide (5 mg/kg), chlorzoxazone (20 mg/kg) and midazolam (10 mg/kg), was orally administration to rats treated twice daily with RA (10, 20 and 40 g/kg) for consecutive 14 days. Blood samples (0.2 mL) were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS/MS. The corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0 (Wenzhou Medical College, Zhejiang, China). Results: In the experiment, there was a statistically significant difference in the t1/2, Cmax, AUC(0-1) and CL for phenacetin and midazolam, while there was no statistical pharmacokinetics difference for tolbutamide and chlorzoxazone. Our study showed that treatment with multiple doses of RA had an inductive effect on rat CYP1A2 and an inhibitory effect on rat CYP3A4 enzyme activity. However, RA has no inductive or inhibitory effect on the activities of CYP2C9 and CYP2E1. Conclusions: Caution is needed when RA is co-administration with some CYP1A2 or CYP3A4 substrates in clinic, because it may result in treatment failure and herb–drug interactions.
CYP, induce, inhibit, RA
Introduction Herbal remedy has been an important part of traditional medicine in China. Alismatis rhizome (RA) (Water Plantain Family, also called ‘‘Zexie’’ in Chinese), one of the commonly used traditional Chinese medicines, is derived from the dried rhizomes of Alisma orientale (Sam.) Juzep. (Alismataceae). It has been used as a diuretic and a folk medicine for diabetes, and it is an important crude drug component for several Chinese preparations, such as Long Dan Xie Gan Wan and Liu Wei Di Huang Wan (Li & Qu, 2012; Xie et al., 2012). For centuries, RA has been prescribed for a variety of disorders including oliguria, edema, gonorrhea with turbid urine or leucorrhea, diarrhea, and dizziness in many Asian countries. Recent studies have also shown that RA has therapeutic effects on various disorders including renal lithiasis (Cao et al., 2004), hypertension (Makino et al., 2002), hepatitis B (Jiang et al., 2006) and nonalcoholic fatty liver disease (Hong et al., 2006). In addition, it was suggested that RA is involved in regulation of inflammation (Han et al., 2013). Despite numerous reports Correspondence: Yao Hou, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China. E-mail: [email protected]. Zhi-sheng Xu, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China. E-mail: [email protected]
History Received 17 September 2013 Revised 21 October 2013 Accepted 6 November 2013 Published online 6 February 2014
on the therapeutic potential of RA, the effect of RA on the cytochrome P450 (CYP) activities is largely unknown. In this study, we sought to uncover a potential inductive or inhibitive function of RA on CYP activities by using rats. CYP are important phase I enzymes in the biotransformation of xenobiotics and their subsequent clearance from the body. The relative abundance and significance of individual CYP enzyme in human drug metabolism varies, the more important CYP isoform families include CYP1A, CYP2C, CYP2E and CYP3A. Induction or inhibition of the P450 enzymes, after exposure to different drugs and chemicals, is directly linked to a number of drug-induced toxicity and drug interactions leading to treatment failure. These drug interactions have become more common as a result of polypharmacy (and/or exposure to environmental pollutants) and the increasing use of alternative medicine, including herbs and natural products (Venkataramanan et al., 2006; Zhou et al., 2003). With the increasing consumption of medicinal herbs, often in combination with or at the same time of conventional therapeutic drugs, it is important to identify constituents in herbal preparations or natural products which may be substrates, inhibitors or inducers of CYPs and thus have the potential to affect the pharmacokinetics of drugs which rely on CYP for their metabolism and clearance.
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Herb–drug interactions have been observed with herbal and natural products when co-administered with warfarin (Bourget et al., 2007; Hu et al., 2005). It is important to investigate drug–herb interactions as well as drug–drug interactions. In vivo experiments concerning drug interactions provide us with information about which drugs might show serious interaction problems, although the results need to be tested in vitro (Lee et al., 2012). Such information can decrease the number of risks from drug–drug and drug–herb interactions. However, few data are available about the effects of RA on substrate drug–enzyme reactions. Probe drugs have been widely used for phenotyping various individual CYP activities, and this approach has been widely used in many investigations in the field of drug metabolism and pharmacogenetics. Probe drugs have been used alone and in combinations, offering necessary benefits. Administration of multiple compounds simultaneously as a ‘‘cocktail’’ strategy, providing no metabolic or analytical interactions from each other, is useful to enhance drug efficiency (Tanaka et al., 2003). Several cocktail combinations have been described including the ‘‘Zientek cocktail’’ (Zientek & Youdim, 2013), ‘‘Lee cocktail’’ (Lee et al., 2013), ‘‘Lin cocktail’’ (Lin et al., 2013), ‘‘Yao cocktail’’ (Yao et al., 2012), ‘‘Oh cocktail’’ (Oh et al., 2012), ‘‘Zhang cocktail’’ (Zhang et al., 2010), ‘‘Liu cocktail’’ (Liu et al., 2009) and others. These methods can minimize intra-subject variability over time. However, the disadvantages of this cocktail method are also well defined: the frequent occurrence of probe drug side-effects, more sample consumption, more time consumption and complicated analytical methods (Tanaka et al., 2003). Nevertheless, the cocktail approach is widely used to assess the influence of herbal medicines on the CYP activities and potential herb–drug interactions (Bao et al., 2012; Han et al., 2012; Su et al., 2013). RA, one of the most important herbal medicines in traditional Chinese medicine, is widely consumed concomitantly with prescribed drugs in Chinese clinics. However, no systematic study has been reported emphasizing the impact of RA on hepatic CYP enzyme activities up to now. With the aim of avoiding possible side effects induced by herb–drug interactions, we evaluated the effects of RA on the activities of CYP1A2, CYP2C9, CYP2E1 and CYP3A4 enzymes in rats. We used a new four-probe drug cocktail (containing phenacetin for CYP1A2, tolbutamide for CYP2C9, chlorzoxazone for CYP2E1 and midazolam for CYP3A4) based on a developed and validated HPLC-MS/MS method to assess CYP activities by comparing pharmacokinetics of the four substrates between control and treatment groups in vivo. We predict that the results may be useful for the clinical safety evaluation of herb–drug interactions involving RA.
Materials and methods Herbal material, chemicals and reagents The dried rhizomes of RA were purchased from Hangzhou Chinese medicine herb tablet factory (Hangzhou, China). Phenacetin (purity498.0%), tolbutamide (purity 498.0%), chlorzoxazone (purity498.0%), midazolam (purity498.0%) and the internal standard carbamazepine (IS, purity498.0%) were also purchased from Sigma-Aldrich
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Company (St. Louis, MO). HPLC grade acetonitrile and methanol were from Merck Company (Darmstadt, Germany). All other chemicals were of analytical grade and used without further purification. Ultra-pure water (resistance418.2 m ) prepared by a Millipore Milli-Q purification system (Bedford, MA). Herbal material processing The crude drug (1 kg) was extracted twice by refluxing with boiling water (1:10, w/v) for 1 h each time. The solution obtained was concentrated under reduced pressure to produce the RA water extract at a concentration equivalent to 4 g of raw material per milliliter, and stored at 4 C until its administration to the rats. Apparatus All analysis was performed with a 1200 Series liquid chromatograph (Agilent Technologies, Waldbronn, Germany) equipped with a quaternary pump, a degasser, an autosampler, a thermostatted column compartment and a Bruker Esquire HCT mass spectrometer (Bruker Technologies, Bremen, Germany) equipped with an electrospray ion source and controlled by ChemStation software (Agilent Technologies, Waldbronn, Germany). Animals Male Sprague–Dawley rats with body weights of 220 30 g were provided by the Animal Care and Use Committee of Wenzhou Medical College. They were housed cages at 23–25 C and allowed free access to regular rodent diet and water. After the 1-week acclimatization period, the rats were used for experiments and all efforts were made to minimize any animal suffering. All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Wenzhou Medical College and were in accordance with the Guide for the Care and Use of Laboratory Animals. Drug administration and sampling Twenty-four male Sprague–Dawley rats were randomly divided into four groups (a total of 24 rats, n ¼ 6): blank control group (BCG), low dosage group (LDG, 10 g/kg), medium dosage group (MDG, 20 g/kg) and high dosage group (HDG, 40 g/kg), which were given the same volume of water or RA by oral administration twice daily. After oral administration for consecutive 14 days, a cocktail solution at a dose of 5 mL/kg, which contained phenacetin (20 mg/kg), tolbutamide (5 mg/kg), chlorzoxazone (20 mg/kg) and midazolam (10 mg/ kg) in CMC-Na solution, was administered orally to all rats in each group. Blood samples (0.2 mL) of each rat were collected pre-dose (0 h) and 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, and 12 h after probe drugs administration through the tail vein and immediately separated by centrifugation at 13 000 rpm for 10 min to obtain plasma. From the seventh blood collection, the rats were treated by oral administration of normal saline of the same blood collection volume in order to restore blood capacity quickly. Plasma samples (100 mL) were transferred to another tube and stored frozen at 80 C until analyzed.
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Table 1. Main pharmacokinetic parameters of phenacetin after multiple doses of RA in rat plasma (n ¼ 6, mean SD). Parameter t1/2 (h) Tmax(h) Cmax (ng/mL) AUC(0–1) (mgh/L) MRT(0–1) (h) CL (L/h/kg)
BCG
LDG
MDG
HDG
6.307 0.455 0.264 0.005 4071.978 231.772 22 720.318 375.609 8.237 0.386 0.980 0.015
5.699 0.568 0.252 0.002 3860.549 183.125 20 847.194 518.087 7.878 0.568 1.063 0.046
5.305 0.740* 0.246 0.004 3644.780 165.036* 16 573.163 508.729* 6.986 0.868* 1.209 0.054*
5.094 0.378* 0.235 0.005* 3444.857 115.921* 14 414.784 458.188** 6.746 0.459* 1.389 0.044**
*Significantly different from control, p50.05. **Significantly different from control, p50.01.
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Sample preparation In a 1.5 mL centrifuge tube, an aliquot of 0.2 mL acetonitrile with carbamazepine (500 ng/mL) as the internal standard was added to 0.1 mL of collected plasma sample. After the tube was vortex-mixed for 1.0 min, the sample was centrifuged at 13 000 g for 10 min. Next, the supernatant (10 mL) was injected into the HPLC-MS/MS system for analysis. Chromatographic conditions Chromatographic separation was achieved on an Agilent Zorbax SB-C18 column (150 mm 2.1 mm, 3.5 mm) with the column temperature set at 30 C. The mobile phase consisted of (A) acetonitrile and (B) 0.1% formic acid in water, and a gradient elution of 10–85% A at 0–1.5 min, 85–85% A at 1.5– 6.0 min, 85–10% A at 6.0–7.0 min and 10–10% A at 7.0– 10.0 min was employed. The flow rate was 0.4 mL/min. The injection volume was 10 mL. The quantification was performed by the peak-area method. The determination of target ions was performed in SIM mode (m/z 180 for phenacetin, m/z 271 for tolbutamide, m/z 168 for chlorzoxazone, m/z 326 for midazolam and m/z 237 for IS) and positive ion electrospray ionization interface. Drying gas flow was set to 6 L/min and temperature to 350 C. Nebuliser pressure and capillary voltage of the system were adjusted to 20 psi and 3500 V, respectively. Statistical analysis The concentration–time profile of each probe drug was analyzed by DAS software (Version 3.0, Wenzhou Medical College, Zhejiang, China) and statistic analyses were tested by t-test using SPSS (Version 13.0, Wenzhou Medical College, Zhejiang, China). A value of p50.05 was considered to be statistically significant.
Results Validation of the cocktail approach Under the conditions described in the experimental section, a reliable liquid chromatography–mass spectrometry has been developed for simultaneous evaluation of the activities of four CYP enzymes (phenacetin for CYP1A2, tolbutamide for CYP2C9, chlorzoxazone for CYP2E1 and midazolam for CYP3A4) in rats. The ion transitions monitored were as follows: m/z 180 for phenacetin, m/z 271 for tolbutamide, m/z 168 for chlorzoxazone, m/z 326 for midazolam and m/z 237 for IS. The lower limit of quantification for phenacetin, tolbutamide, chlorzoxazone and midazolam were all
Figure 1. Mean plasma concentration–time curves of phenacetin in rats.
5.0 ng/mL, and the assay ranges used were all 5.0–1000 ng/ mL in rat plasma. The plasma samples with analyte concentration above the upper limit of quantitation were diluted with blank rat plasma. Correlation coefficient of the calibration curves for phenacetin was 0.9977, for tolbutamide 0.9973, for chlorzoxazone 0.9983 and for midazolam 0.9976. The intraday and inter-day CVs for the low-, medium- and high-quality control samples were less than 15%. The result of the chromatographic validation showed that assay methods were suitable for this study. Effect of RA on rat CYP1A2 The effects of different treatment groups of RA on pharmacokinetic parameters of phenacetin in rats are presented in Table 1. Mean plasma concentration–time curves of phenacetin in different groups are presented in Figure 1. After pretreatment with RA, the t1/2, Cmax, AUC(0–1) and MRT(0–1) of phenacetin in MDG were decreased significantly by 15.9, 10.5, 27.1 and 15.2% compared to those of BCG, CL of phenacetin in MDG was increased significantly by 23.3%. The t1/2, Tmax, Cmax, AUC(0–1) and MRT(0–1) of phenacetin in HDG were decreased significantly by 19.2, 11.0, 15.4, 36.6 and 18.1% compared to those of BCG, CL of phenacetin in HDG was increased significantly by 41.7%. The pharmacokinetic parameters of phenacetin in rats showed no significant difference between LDG and BCG; however, these parameters were altered for phenacetin. The results indicated that metabolism of phenacetin in these treatment groups was evidently speeded up, and RA had the potential to induce rat hepatic CYP1A2 activity in vivo.
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Table 2. Main pharmacokinetic parameters of tolbutamide after multiple doses of RA in rat plasma (n ¼ 6, mean SD). Parameter
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t1/2 (h) Tmax(h) Cmax (ng/mL) AUC(0–1) (mgh/L) MRT(0–1) (h) CL (L/h/kg)
BCG
LDG
MDG
HDG
6.025 0.525 2.529 0.189 16 550.573 433.216 186 920.019 2039.827 9.699 1.914 0.027 0.003
5.973 0.624 2.571 0.535 16 312.249 363.386 193 030.113 2715.062 9.637 1.536 0.026 0.004
5.703 0.914 2.602 0.454 16 749.571 247.383 189 093.764 2173.525 9.676 1.065 0.027 0.002
5.913 0.800 2.486 0.488 16 346.857 262.406 188 684.453 2525.788 9.640 1.052 0.026 0.002
Figure 2. Mean plasma concentration–time curves of tolbutamide in rats.
Effect of RA on rat CYP2C9 The effects of different treatment groups of RA on pharmacokinetic parameters of tolbutamide in rats are presented in Table 2. Mean plasma concentration–time curves of tolbutamide in different groups are presented in Figure 2. After pretreatment with RA, the pharmacokinetic parameters (t1/2, Tmax, Cmax, AUC(0–1), MRT(0–1) and CL) of tolbutamide in rats showed no significant difference between different treatment groups and BCG. The results indicated that RA showed no influence on rat hepatic CYP2C9 activity in vivo. Effect of RA on rat CYP2E1 The effects of different treatment groups of RA on pharmacokinetic parameters of chlorzoxazone in rats are presented in Table 3. Mean plasma concentration–time curves of chlorzoxazone in different groups are presented in Figure 3. The pharmacokinetic parameters of chlorzoxazone in rats showed no significant difference between different treatment groups and BCG. No apparent influences were observed on the pharmacokinetic parameters in these treatment groups. The results also indicated that RA showed no influence on rat hepatic CYP2E1 activity in vivo. Effect of RA on rat CYP3A4 The effects of different treatment groups of RA on pharmacokinetic parameters of midazolam in rats are presented in Table 4. Mean plasma concentration–time curves
of midazolam in different groups are presented in Figure 4. After pretreatment with RA, the AUC(0–1) of midazolam in LDG were increased significantly by 12.7% compared to those of BCG, CL of midazolam in LDG was decreased significantly by 11.0%. Meanwhile, the AUC(0–1) and MRT(0–1) of midazolam in MDG were increased significantly by 30.0% and 10.5% compared to those of BCG, CL of midazolam in MDG was decreased significantly by 22.8%. Moreover, the t1/2, Tmax, Cmax, AUC(0–1) and MRT(0–1) of midazolam in HDG were increased significantly by 25.4, 20.6, 12.9, 60.1 and 29.8% compared to those of BCG, CL of midazolam in HDG was decreased significantly by 37.4%. No apparent influences were observed on other pharmacokinetic parameters in these treatment groups. The results indicated that metabolism of midazolam in these treatment groups was evidently slowed down, and RA had the potential to inhibit rat hepatic CYP3A4 activity in vivo.
Discussion People have used medicinal herbs since ancient times and herbs still play a significant role as an essential part of human medicine in many countries and cultures. Traditional herbal medicine uses herbs such as bitter orange, ginseng and ginkgo for curing various health conditions. However, there are various adverse effects associated with using herbal medicines. Most reports of such adverse effects relate to Chinese herbal medicines. Specifically, the adverse effects of Chinese herbal medicines are classified into three types: type A, acute and predictable reactions that are extensions of the pharmacological effects and generally dose dependent; type B, idiosyncratic reactions that are not predicted by pharmacology, are not related to dose, and can cause significant death; and type C, cumulative, chronic or delayed toxicity (Edwards & Aronson, 2000). In addition to these reported toxicities of using herbal medicines alone, various problems have been reported using herbal medicines in combination with drugs. For example, various herbal products have the potential to inhibit or induce human CYP enzymes following the concomitant intake of drugs with herbal extracts (Foster et al., 2003). St. John’s Wort (Hypericum sp., Hypericaceae) is effective for treating mild to moderate depression and is a powerful inducer of CYP3A4; consequently, it accelerates the clearance of many prescription drugs including alprazolam (Markowitz et al., 2003). In our study, a developed and validated HPLC-MS/MS method was used to determine the levels of the four probe drugs (phenacetin for CYP1A2, tolbutamide for CYP2C9, chlorzoxazone for CYP2E1 and midazolam for CYP3A4) in
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Table 3. Main pharmacokinetic parameters of chlorzoxazone after multiple doses of RA in rat plasma (n ¼ 6, mean SD). Parameter
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t1/2 (h) Tmax (h) Cmax (ng/mL) AUC(0–1) (mgh/L) MRT(0–1) (h) CL (L/h/kg)
BCG
LDG
MDG
HDG
4.819 0.159 3.101 0.298 9940.143 155.694 90 333.422 1480.638 7.759 0.208 0.219 0.004
4.670 0.156 3.043 0.434 9809.714 170.832 88 338.849 1520.275 7.520 0.168 0.217 0.001
4.798 0.117 2.987 0.278 9777.571 168.588 89 129.107 1261.451 7.671 0.148 0.218 0.004
4.689 0.144 3.143 0.378 9750.857 164.919 90 609.608 1748.562 7.797 0.199 0.217 0.004
Figure 3. Mean plasma concentration–time curves of chlorzoxazone in rats.
rat plasma after oral administration of RA for 14 days. This method is available to measure the enzyme activity of CYPs. The probe substrate approach can be used as a general screening tool to characterize CYPs’ inhibition and induction effects for a drug. It can not only detect the activity of a variety of drug-metabolizing enzymes but also get the inductive or inhibitory effect by the change of the activity of the one enzyme. The cocktail approach has been, in general, proposed as a screening tool for potential in vivo drug–drug interactions. In our present study, the potential effect of RA on CYP activities including CYP1A2, CYP2C9, CYP2E1 and CYP3A4 in rats was assessed. And it is also providing the opportunity to help us to find the metabolites of RA. In this study, t1/2 and AUC(0–1) of phenacetin in HDG significantly decreased, and the corresponding CL markedly increased compared to BCG. In addition, the pharmacokinetic parameters of phenacetin in rats showed no significant difference among LDG, MDG and BCG; however, these parameters were altered for phenacetin. Overall, it illustrated that the activity of CYP1A2 tended to be induced. However, according to our results, we find that AUC(0–1) and t1/2 of midazolam in different treatment groups significantly increased, and the corresponding CL markedly decreased, which illustrated that the activity of CYP3A4 tended to be inhibited. The complicated mechanisms of the effect of RA on the activities of CYP1A2 and CYP3A4 remain to be further studied. Another possibility is that the concentration of RA in our study might not be high enough to inhibit or
induce the activities of CYP1A2 and CYP3A4, respectively. The study focusing on the effect of RA on the CYP enzymes in vitro is so critical that it helps us to make the mechanism of the metabolism of RA understood completely. So, in vitro study of RA impacts on CYP1A2 and CYP3A4 activities needs to be discussed in the future. Moreover, the dose dependency and kinetics of RA on the activities of CYP1A2 and CYP3A4 in vitro also remain to be studied. Finally, we find that RA did not influence the plasma concentration of another two probe drugs (tolbutamide and chlorzoxazone) as good as their associated pharmacokinetic parameters, and had very little or no affect on the CYP activities of CYP2C9 and CYP2E1. It is well known that a major contributing factor of the drug–drug interaction is the inhibition of CYP enzymemediated activities, of which human CYP1A2 accounts for about 13% of the total CYP content in human liver (Shimada et al., 1994). CYP1A2 is mainly responsible for metabolizing a variety of clinically important drugs, such as clozapine, ropivacaine, olanzapine and theophylline. In addition to this, CYP1A2 also metabolizes a number of procarcinogens and endogenous substrates (Zhou et al., 2009). RA can increase the activity of CYP1A2 in this study. It suggests that RA helps restore hepatic drug metabolism, benefits body discharging exogenous materials and endogenous products and helps to explain the antitoxic mechanism of itself. CYP3A4 is the most abundant CYP found in human liver (40%) metabolizing more than 50% clinically prescribed drugs. Many cancer chemotherapeutic agents such as vinca alkaloids, paclitaxel, irinotecan and other widely used drugs such as amiodarone, diltiazem, lovastatin and midazolam are its substrates (Zhou et al., 2009). The inhibition on CYP3A4 could be highly important for many conditions, which could be of concern, such as the endogenous metabolism, specific physiological functions, the biotransformation of ubiquitous environmental pollutants and changes in the pharmacokinetics of coadministered drugs. With the great use of RA as a herbal medicine, people should pay more attention to its side effects caused by drug–drug interactions when they are administrated with other drugs, especially with substrates of CYP3A4.
Conclusion In conclusion, the inconspicuous effects of RA in vivo on probes of CYP2C9 and CYP2E1 metabolism suggest that there is no clinically relevant drug–drug interaction between the drugs metabolized by these enzymes and RA when
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Table 4. Main pharmacokinetic parameters of midazolam after multiple doses of RA in rat plasma (n ¼ 6, mean SD). Parameter t1/2 (h) Tmax(h) Cmax (ng/mL) AUC(0–1) (mgh/L) MRT(0–1) (h) CL (L/h/kg)
BCG
LDG
MDG
HDG
5.365 0.191 0.247 0.004 6256.714 127.088 39 312.245 1602.686 7.464 0.588 0.254 0.004
5.717 0.250 0.266 0.005 6554.429 152.446 44 292.342 1159.704* 8.024 0.652 0.226 0.006*
5.739 0.168 0.274 0.005 6768.429 149.608 51 103.299 2184.750* 8.246 0.927* 0.196 0.003*
6.728 0.207* 0.298 0.004* 7063.143 182.872* 62 943.242 2433.412** 9.686 0.637* 0.159 0.003**
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*Significantly different from control, p50.05. **Significantly different from control, p50.01.
Figure 4. Mean plasma concentration–time curves of midazolam in rats.
they are used concomitantly. However, from our present results, we cannot exclude that comedication of RA with drugs metabolized by human CYP1A2 and CYP3A4 may induce or inhibit metabolism of these drugs and change plasma concentrations of these drugs, which will result in relevant drug–drug interaction. Further clinical studies are required to fully assess the safety of RA in terms of CYP1A2 induction and CYP3A4 inhibition.
Declaration of interest The authors report no conflicts of interest.
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Han YL, Li D, Ren B, et al. (2012). Evaluation of impact of Herba Erigerontis injection, a Chinese herbal prescription, on rat hepatic cytochrome P450 enzymes by cocktail probe drugs. J Ethnopharmacol 139:104–9. Hong X, Tang H, Wu L, Li L. (2006). Protective effects of the Alisma orientalis extract on the experimental nonalcoholic fatty liver disease. J Pharm Pharmacol 58:1391–8. Hu Z, Yang X, Ho PC, et al. (2005). Herb-drug interactions: A literature review. Drugs 65:1239–82. Jiang ZY, Zhang XM, Zhang FX, et al. (2006). A new triterpene and anti-hepatitis B virus active compounds from Alisma orientalis. Planta Med 72:951–4. Lee JT, Pao LH, Hsiong CH, et al. (2013). Validated liquid chromatography–tandem mass spectrometry method for determination of totally nine probe metabolites of cytochrome P450 enzymes and UDP-glucuronosyltransferases. Talanta 106:220–8. Lee M, Park J, Lim MS, et al. (2012). High-throughput screening of inhibitory effects of Bo-yang-hwan-o-tang on human cytochrome P450 isoforms in vitro using UPLC/MS/MS. Anal Sci 28:1197–201. Li Q, Qu H. (2012). Study on the hypoglycemic activities and metabolism of alcohol extract of Alismatis Rhizoma. Fitoterapia 83: 1046–53. Lin W, Zhang J, Ling X, et al. (2013). Evaluation of the effect of TM208 on the activity of five cytochrome P450 enzymes using on-line solidphase extraction HPLC-DAD: A cocktail approach. J Chromatogr B: Analyt Technol Biomed Life Sci 923–4:29–36. Liu Y, Jiao J, Zhang C, Lou J. (2009). A simplified method to determine five cytochrome p450 probe drugs by HPLC in a single run. Biol Pharm Bull 32:717–20. Makino B, Kobayashi M, Kimura K, et al. (2002). Local variation in the content of angiotensin II and arginine vasopressin receptor antagonistic terpenoids in the rhizomes of Alisma orientale. Planta Med 68:226–31. Markowitz JS, Donovan JL, Devane CL, et al. (2003). Effect of St John’s wort on drug metabolism by induction of cytochrome P450 3A4 enzyme. JAMA 290:1500–4. Oh KS, Park SJ, Shinde DD, et al. (2012). High-sensitivity liquid chromatography–tandem mass spectrometry for the simultaneous determination of five drugs and their cytochrome P450-specific probe metabolites in human plasma. J Chromatogr B: Anal Technol Biomed Life Sci 895:56–64. Shimada T, Yamazaki H, Mimura M, et al. (1994). Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: Studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 270:414–23. Su T, Mao C, Yin F, et al. (2013). Effects of unprocessed versus vinegarprocessed Schisandra chinensis on the activity and mRNA expression of CYP1A2, CYP2E1 and CYP3A4 enzymes in rats. J Ethnopharmacol 146:734–43. Tanaka E, Kurata N, Yasuhara H. (2003). How useful is the ‘‘cocktail approach’’ for evaluating human hepatic drug metabolizing capacity using cytochrome P450 phenotyping probes in vivo? J Clin Pharm Ther 28:157–65 Venkataramanan R, Komoroski B, Strom S. (2006). In vitro and in vivo assessment of herb drug interactions. Life Sci 78:2105–15. Xie W, Zhao Y, Du L. (2012). Emerging approaches of traditional Chinese medicine formulas for the treatment of hyperlipidemia. J Ethnopharmacol 140:345–67.
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ORIGINAL ARTICLE
Effects of Alismatis rhizome on rat cytochrome P450 enzymes Yi Huang1, Shuang-li Zheng2, Zhi-sheng Xu3, and Yao Hou4
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1
Department of General Surgery of Wenzhou People’s Hospital, Wenzhou, China, 2Department of Pediatric Surgery of the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, China, 3Department of Hematology of the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, China, and 4Department of Ultrasound Imaging of the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China
Abstract
Keywords
Context: Alismatis rhizome (RA) (Water Plantain Family, also called ‘‘Zexie’’ in Chinese), one of the commonly used components of traditional Chinese medicines, is derived from the dried rhizomes of Alisma orientalis (Sam.) Juzep. (Alismataceae). Objective: This study explores the RA influences on rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9, CYP2E1 and CYP3A4) by using cocktail probe drugs in vivo. Materials and methods: A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (20 mg/kg), tolbutamide (5 mg/kg), chlorzoxazone (20 mg/kg) and midazolam (10 mg/kg), was orally administration to rats treated twice daily with RA (10, 20 and 40 g/kg) for consecutive 14 days. Blood samples (0.2 mL) were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS/MS. The corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0 (Wenzhou Medical College, Zhejiang, China). Results: In the experiment, there was a statistically significant difference in the t1/2, Cmax, AUC(0-1) and CL for phenacetin and midazolam, while there was no statistical pharmacokinetics difference for tolbutamide and chlorzoxazone. Our study showed that treatment with multiple doses of RA had an inductive effect on rat CYP1A2 and an inhibitory effect on rat CYP3A4 enzyme activity. However, RA has no inductive or inhibitory effect on the activities of CYP2C9 and CYP2E1. Conclusions: Caution is needed when RA is co-administration with some CYP1A2 or CYP3A4 substrates in clinic, because it may result in treatment failure and herb–drug interactions.
CYP, induce, inhibit, RA
Introduction Herbal remedy has been an important part of traditional medicine in China. Alismatis rhizome (RA) (Water Plantain Family, also called ‘‘Zexie’’ in Chinese), one of the commonly used traditional Chinese medicines, is derived from the dried rhizomes of Alisma orientale (Sam.) Juzep. (Alismataceae). It has been used as a diuretic and a folk medicine for diabetes, and it is an important crude drug component for several Chinese preparations, such as Long Dan Xie Gan Wan and Liu Wei Di Huang Wan (Li & Qu, 2012; Xie et al., 2012). For centuries, RA has been prescribed for a variety of disorders including oliguria, edema, gonorrhea with turbid urine or leucorrhea, diarrhea, and dizziness in many Asian countries. Recent studies have also shown that RA has therapeutic effects on various disorders including renal lithiasis (Cao et al., 2004), hypertension (Makino et al., 2002), hepatitis B (Jiang et al., 2006) and nonalcoholic fatty liver disease (Hong et al., 2006). In addition, it was suggested that RA is involved in regulation of inflammation (Han et al., 2013). Despite numerous reports Correspondence: Yao Hou, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China. E-mail: [email protected]. Zhi-sheng Xu, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China. E-mail: [email protected]
History Received 17 September 2013 Revised 21 October 2013 Accepted 6 November 2013 Published online 6 February 2014
on the therapeutic potential of RA, the effect of RA on the cytochrome P450 (CYP) activities is largely unknown. In this study, we sought to uncover a potential inductive or inhibitive function of RA on CYP activities by using rats. CYP are important phase I enzymes in the biotransformation of xenobiotics and their subsequent clearance from the body. The relative abundance and significance of individual CYP enzyme in human drug metabolism varies, the more important CYP isoform families include CYP1A, CYP2C, CYP2E and CYP3A. Induction or inhibition of the P450 enzymes, after exposure to different drugs and chemicals, is directly linked to a number of drug-induced toxicity and drug interactions leading to treatment failure. These drug interactions have become more common as a result of polypharmacy (and/or exposure to environmental pollutants) and the increasing use of alternative medicine, including herbs and natural products (Venkataramanan et al., 2006; Zhou et al., 2003). With the increasing consumption of medicinal herbs, often in combination with or at the same time of conventional therapeutic drugs, it is important to identify constituents in herbal preparations or natural products which may be substrates, inhibitors or inducers of CYPs and thus have the potential to affect the pharmacokinetics of drugs which rely on CYP for their metabolism and clearance.
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Herb–drug interactions have been observed with herbal and natural products when co-administered with warfarin (Bourget et al., 2007; Hu et al., 2005). It is important to investigate drug–herb interactions as well as drug–drug interactions. In vivo experiments concerning drug interactions provide us with information about which drugs might show serious interaction problems, although the results need to be tested in vitro (Lee et al., 2012). Such information can decrease the number of risks from drug–drug and drug–herb interactions. However, few data are available about the effects of RA on substrate drug–enzyme reactions. Probe drugs have been widely used for phenotyping various individual CYP activities, and this approach has been widely used in many investigations in the field of drug metabolism and pharmacogenetics. Probe drugs have been used alone and in combinations, offering necessary benefits. Administration of multiple compounds simultaneously as a ‘‘cocktail’’ strategy, providing no metabolic or analytical interactions from each other, is useful to enhance drug efficiency (Tanaka et al., 2003). Several cocktail combinations have been described including the ‘‘Zientek cocktail’’ (Zientek & Youdim, 2013), ‘‘Lee cocktail’’ (Lee et al., 2013), ‘‘Lin cocktail’’ (Lin et al., 2013), ‘‘Yao cocktail’’ (Yao et al., 2012), ‘‘Oh cocktail’’ (Oh et al., 2012), ‘‘Zhang cocktail’’ (Zhang et al., 2010), ‘‘Liu cocktail’’ (Liu et al., 2009) and others. These methods can minimize intra-subject variability over time. However, the disadvantages of this cocktail method are also well defined: the frequent occurrence of probe drug side-effects, more sample consumption, more time consumption and complicated analytical methods (Tanaka et al., 2003). Nevertheless, the cocktail approach is widely used to assess the influence of herbal medicines on the CYP activities and potential herb–drug interactions (Bao et al., 2012; Han et al., 2012; Su et al., 2013). RA, one of the most important herbal medicines in traditional Chinese medicine, is widely consumed concomitantly with prescribed drugs in Chinese clinics. However, no systematic study has been reported emphasizing the impact of RA on hepatic CYP enzyme activities up to now. With the aim of avoiding possible side effects induced by herb–drug interactions, we evaluated the effects of RA on the activities of CYP1A2, CYP2C9, CYP2E1 and CYP3A4 enzymes in rats. We used a new four-probe drug cocktail (containing phenacetin for CYP1A2, tolbutamide for CYP2C9, chlorzoxazone for CYP2E1 and midazolam for CYP3A4) based on a developed and validated HPLC-MS/MS method to assess CYP activities by comparing pharmacokinetics of the four substrates between control and treatment groups in vivo. We predict that the results may be useful for the clinical safety evaluation of herb–drug interactions involving RA.
Materials and methods Herbal material, chemicals and reagents The dried rhizomes of RA were purchased from Hangzhou Chinese medicine herb tablet factory (Hangzhou, China). Phenacetin (purity498.0%), tolbutamide (purity 498.0%), chlorzoxazone (purity498.0%), midazolam (purity498.0%) and the internal standard carbamazepine (IS, purity498.0%) were also purchased from Sigma-Aldrich
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Company (St. Louis, MO). HPLC grade acetonitrile and methanol were from Merck Company (Darmstadt, Germany). All other chemicals were of analytical grade and used without further purification. Ultra-pure water (resistance418.2 m ) prepared by a Millipore Milli-Q purification system (Bedford, MA). Herbal material processing The crude drug (1 kg) was extracted twice by refluxing with boiling water (1:10, w/v) for 1 h each time. The solution obtained was concentrated under reduced pressure to produce the RA water extract at a concentration equivalent to 4 g of raw material per milliliter, and stored at 4 C until its administration to the rats. Apparatus All analysis was performed with a 1200 Series liquid chromatograph (Agilent Technologies, Waldbronn, Germany) equipped with a quaternary pump, a degasser, an autosampler, a thermostatted column compartment and a Bruker Esquire HCT mass spectrometer (Bruker Technologies, Bremen, Germany) equipped with an electrospray ion source and controlled by ChemStation software (Agilent Technologies, Waldbronn, Germany). Animals Male Sprague–Dawley rats with body weights of 220 30 g were provided by the Animal Care and Use Committee of Wenzhou Medical College. They were housed cages at 23–25 C and allowed free access to regular rodent diet and water. After the 1-week acclimatization period, the rats were used for experiments and all efforts were made to minimize any animal suffering. All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Wenzhou Medical College and were in accordance with the Guide for the Care and Use of Laboratory Animals. Drug administration and sampling Twenty-four male Sprague–Dawley rats were randomly divided into four groups (a total of 24 rats, n ¼ 6): blank control group (BCG), low dosage group (LDG, 10 g/kg), medium dosage group (MDG, 20 g/kg) and high dosage group (HDG, 40 g/kg), which were given the same volume of water or RA by oral administration twice daily. After oral administration for consecutive 14 days, a cocktail solution at a dose of 5 mL/kg, which contained phenacetin (20 mg/kg), tolbutamide (5 mg/kg), chlorzoxazone (20 mg/kg) and midazolam (10 mg/ kg) in CMC-Na solution, was administered orally to all rats in each group. Blood samples (0.2 mL) of each rat were collected pre-dose (0 h) and 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, and 12 h after probe drugs administration through the tail vein and immediately separated by centrifugation at 13 000 rpm for 10 min to obtain plasma. From the seventh blood collection, the rats were treated by oral administration of normal saline of the same blood collection volume in order to restore blood capacity quickly. Plasma samples (100 mL) were transferred to another tube and stored frozen at 80 C until analyzed.
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Table 1. Main pharmacokinetic parameters of phenacetin after multiple doses of RA in rat plasma (n ¼ 6, mean SD). Parameter t1/2 (h) Tmax(h) Cmax (ng/mL) AUC(0–1) (mgh/L) MRT(0–1) (h) CL (L/h/kg)
BCG
LDG
MDG
HDG
6.307 0.455 0.264 0.005 4071.978 231.772 22 720.318 375.609 8.237 0.386 0.980 0.015
5.699 0.568 0.252 0.002 3860.549 183.125 20 847.194 518.087 7.878 0.568 1.063 0.046
5.305 0.740* 0.246 0.004 3644.780 165.036* 16 573.163 508.729* 6.986 0.868* 1.209 0.054*
5.094 0.378* 0.235 0.005* 3444.857 115.921* 14 414.784 458.188** 6.746 0.459* 1.389 0.044**
*Significantly different from control, p50.05. **Significantly different from control, p50.01.
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Sample preparation In a 1.5 mL centrifuge tube, an aliquot of 0.2 mL acetonitrile with carbamazepine (500 ng/mL) as the internal standard was added to 0.1 mL of collected plasma sample. After the tube was vortex-mixed for 1.0 min, the sample was centrifuged at 13 000 g for 10 min. Next, the supernatant (10 mL) was injected into the HPLC-MS/MS system for analysis. Chromatographic conditions Chromatographic separation was achieved on an Agilent Zorbax SB-C18 column (150 mm 2.1 mm, 3.5 mm) with the column temperature set at 30 C. The mobile phase consisted of (A) acetonitrile and (B) 0.1% formic acid in water, and a gradient elution of 10–85% A at 0–1.5 min, 85–85% A at 1.5– 6.0 min, 85–10% A at 6.0–7.0 min and 10–10% A at 7.0– 10.0 min was employed. The flow rate was 0.4 mL/min. The injection volume was 10 mL. The quantification was performed by the peak-area method. The determination of target ions was performed in SIM mode (m/z 180 for phenacetin, m/z 271 for tolbutamide, m/z 168 for chlorzoxazone, m/z 326 for midazolam and m/z 237 for IS) and positive ion electrospray ionization interface. Drying gas flow was set to 6 L/min and temperature to 350 C. Nebuliser pressure and capillary voltage of the system were adjusted to 20 psi and 3500 V, respectively. Statistical analysis The concentration–time profile of each probe drug was analyzed by DAS software (Version 3.0, Wenzhou Medical College, Zhejiang, China) and statistic analyses were tested by t-test using SPSS (Version 13.0, Wenzhou Medical College, Zhejiang, China). A value of p50.05 was considered to be statistically significant.
Results Validation of the cocktail approach Under the conditions described in the experimental section, a reliable liquid chromatography–mass spectrometry has been developed for simultaneous evaluation of the activities of four CYP enzymes (phenacetin for CYP1A2, tolbutamide for CYP2C9, chlorzoxazone for CYP2E1 and midazolam for CYP3A4) in rats. The ion transitions monitored were as follows: m/z 180 for phenacetin, m/z 271 for tolbutamide, m/z 168 for chlorzoxazone, m/z 326 for midazolam and m/z 237 for IS. The lower limit of quantification for phenacetin, tolbutamide, chlorzoxazone and midazolam were all
Figure 1. Mean plasma concentration–time curves of phenacetin in rats.
5.0 ng/mL, and the assay ranges used were all 5.0–1000 ng/ mL in rat plasma. The plasma samples with analyte concentration above the upper limit of quantitation were diluted with blank rat plasma. Correlation coefficient of the calibration curves for phenacetin was 0.9977, for tolbutamide 0.9973, for chlorzoxazone 0.9983 and for midazolam 0.9976. The intraday and inter-day CVs for the low-, medium- and high-quality control samples were less than 15%. The result of the chromatographic validation showed that assay methods were suitable for this study. Effect of RA on rat CYP1A2 The effects of different treatment groups of RA on pharmacokinetic parameters of phenacetin in rats are presented in Table 1. Mean plasma concentration–time curves of phenacetin in different groups are presented in Figure 1. After pretreatment with RA, the t1/2, Cmax, AUC(0–1) and MRT(0–1) of phenacetin in MDG were decreased significantly by 15.9, 10.5, 27.1 and 15.2% compared to those of BCG, CL of phenacetin in MDG was increased significantly by 23.3%. The t1/2, Tmax, Cmax, AUC(0–1) and MRT(0–1) of phenacetin in HDG were decreased significantly by 19.2, 11.0, 15.4, 36.6 and 18.1% compared to those of BCG, CL of phenacetin in HDG was increased significantly by 41.7%. The pharmacokinetic parameters of phenacetin in rats showed no significant difference between LDG and BCG; however, these parameters were altered for phenacetin. The results indicated that metabolism of phenacetin in these treatment groups was evidently speeded up, and RA had the potential to induce rat hepatic CYP1A2 activity in vivo.
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Table 2. Main pharmacokinetic parameters of tolbutamide after multiple doses of RA in rat plasma (n ¼ 6, mean SD). Parameter
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t1/2 (h) Tmax(h) Cmax (ng/mL) AUC(0–1) (mgh/L) MRT(0–1) (h) CL (L/h/kg)
BCG
LDG
MDG
HDG
6.025 0.525 2.529 0.189 16 550.573 433.216 186 920.019 2039.827 9.699 1.914 0.027 0.003
5.973 0.624 2.571 0.535 16 312.249 363.386 193 030.113 2715.062 9.637 1.536 0.026 0.004
5.703 0.914 2.602 0.454 16 749.571 247.383 189 093.764 2173.525 9.676 1.065 0.027 0.002
5.913 0.800 2.486 0.488 16 346.857 262.406 188 684.453 2525.788 9.640 1.052 0.026 0.002
Figure 2. Mean plasma concentration–time curves of tolbutamide in rats.
Effect of RA on rat CYP2C9 The effects of different treatment groups of RA on pharmacokinetic parameters of tolbutamide in rats are presented in Table 2. Mean plasma concentration–time curves of tolbutamide in different groups are presented in Figure 2. After pretreatment with RA, the pharmacokinetic parameters (t1/2, Tmax, Cmax, AUC(0–1), MRT(0–1) and CL) of tolbutamide in rats showed no significant difference between different treatment groups and BCG. The results indicated that RA showed no influence on rat hepatic CYP2C9 activity in vivo. Effect of RA on rat CYP2E1 The effects of different treatment groups of RA on pharmacokinetic parameters of chlorzoxazone in rats are presented in Table 3. Mean plasma concentration–time curves of chlorzoxazone in different groups are presented in Figure 3. The pharmacokinetic parameters of chlorzoxazone in rats showed no significant difference between different treatment groups and BCG. No apparent influences were observed on the pharmacokinetic parameters in these treatment groups. The results also indicated that RA showed no influence on rat hepatic CYP2E1 activity in vivo. Effect of RA on rat CYP3A4 The effects of different treatment groups of RA on pharmacokinetic parameters of midazolam in rats are presented in Table 4. Mean plasma concentration–time curves
of midazolam in different groups are presented in Figure 4. After pretreatment with RA, the AUC(0–1) of midazolam in LDG were increased significantly by 12.7% compared to those of BCG, CL of midazolam in LDG was decreased significantly by 11.0%. Meanwhile, the AUC(0–1) and MRT(0–1) of midazolam in MDG were increased significantly by 30.0% and 10.5% compared to those of BCG, CL of midazolam in MDG was decreased significantly by 22.8%. Moreover, the t1/2, Tmax, Cmax, AUC(0–1) and MRT(0–1) of midazolam in HDG were increased significantly by 25.4, 20.6, 12.9, 60.1 and 29.8% compared to those of BCG, CL of midazolam in HDG was decreased significantly by 37.4%. No apparent influences were observed on other pharmacokinetic parameters in these treatment groups. The results indicated that metabolism of midazolam in these treatment groups was evidently slowed down, and RA had the potential to inhibit rat hepatic CYP3A4 activity in vivo.
Discussion People have used medicinal herbs since ancient times and herbs still play a significant role as an essential part of human medicine in many countries and cultures. Traditional herbal medicine uses herbs such as bitter orange, ginseng and ginkgo for curing various health conditions. However, there are various adverse effects associated with using herbal medicines. Most reports of such adverse effects relate to Chinese herbal medicines. Specifically, the adverse effects of Chinese herbal medicines are classified into three types: type A, acute and predictable reactions that are extensions of the pharmacological effects and generally dose dependent; type B, idiosyncratic reactions that are not predicted by pharmacology, are not related to dose, and can cause significant death; and type C, cumulative, chronic or delayed toxicity (Edwards & Aronson, 2000). In addition to these reported toxicities of using herbal medicines alone, various problems have been reported using herbal medicines in combination with drugs. For example, various herbal products have the potential to inhibit or induce human CYP enzymes following the concomitant intake of drugs with herbal extracts (Foster et al., 2003). St. John’s Wort (Hypericum sp., Hypericaceae) is effective for treating mild to moderate depression and is a powerful inducer of CYP3A4; consequently, it accelerates the clearance of many prescription drugs including alprazolam (Markowitz et al., 2003). In our study, a developed and validated HPLC-MS/MS method was used to determine the levels of the four probe drugs (phenacetin for CYP1A2, tolbutamide for CYP2C9, chlorzoxazone for CYP2E1 and midazolam for CYP3A4) in
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Table 3. Main pharmacokinetic parameters of chlorzoxazone after multiple doses of RA in rat plasma (n ¼ 6, mean SD). Parameter
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t1/2 (h) Tmax (h) Cmax (ng/mL) AUC(0–1) (mgh/L) MRT(0–1) (h) CL (L/h/kg)
BCG
LDG
MDG
HDG
4.819 0.159 3.101 0.298 9940.143 155.694 90 333.422 1480.638 7.759 0.208 0.219 0.004
4.670 0.156 3.043 0.434 9809.714 170.832 88 338.849 1520.275 7.520 0.168 0.217 0.001
4.798 0.117 2.987 0.278 9777.571 168.588 89 129.107 1261.451 7.671 0.148 0.218 0.004
4.689 0.144 3.143 0.378 9750.857 164.919 90 609.608 1748.562 7.797 0.199 0.217 0.004
Figure 3. Mean plasma concentration–time curves of chlorzoxazone in rats.
rat plasma after oral administration of RA for 14 days. This method is available to measure the enzyme activity of CYPs. The probe substrate approach can be used as a general screening tool to characterize CYPs’ inhibition and induction effects for a drug. It can not only detect the activity of a variety of drug-metabolizing enzymes but also get the inductive or inhibitory effect by the change of the activity of the one enzyme. The cocktail approach has been, in general, proposed as a screening tool for potential in vivo drug–drug interactions. In our present study, the potential effect of RA on CYP activities including CYP1A2, CYP2C9, CYP2E1 and CYP3A4 in rats was assessed. And it is also providing the opportunity to help us to find the metabolites of RA. In this study, t1/2 and AUC(0–1) of phenacetin in HDG significantly decreased, and the corresponding CL markedly increased compared to BCG. In addition, the pharmacokinetic parameters of phenacetin in rats showed no significant difference among LDG, MDG and BCG; however, these parameters were altered for phenacetin. Overall, it illustrated that the activity of CYP1A2 tended to be induced. However, according to our results, we find that AUC(0–1) and t1/2 of midazolam in different treatment groups significantly increased, and the corresponding CL markedly decreased, which illustrated that the activity of CYP3A4 tended to be inhibited. The complicated mechanisms of the effect of RA on the activities of CYP1A2 and CYP3A4 remain to be further studied. Another possibility is that the concentration of RA in our study might not be high enough to inhibit or
induce the activities of CYP1A2 and CYP3A4, respectively. The study focusing on the effect of RA on the CYP enzymes in vitro is so critical that it helps us to make the mechanism of the metabolism of RA understood completely. So, in vitro study of RA impacts on CYP1A2 and CYP3A4 activities needs to be discussed in the future. Moreover, the dose dependency and kinetics of RA on the activities of CYP1A2 and CYP3A4 in vitro also remain to be studied. Finally, we find that RA did not influence the plasma concentration of another two probe drugs (tolbutamide and chlorzoxazone) as good as their associated pharmacokinetic parameters, and had very little or no affect on the CYP activities of CYP2C9 and CYP2E1. It is well known that a major contributing factor of the drug–drug interaction is the inhibition of CYP enzymemediated activities, of which human CYP1A2 accounts for about 13% of the total CYP content in human liver (Shimada et al., 1994). CYP1A2 is mainly responsible for metabolizing a variety of clinically important drugs, such as clozapine, ropivacaine, olanzapine and theophylline. In addition to this, CYP1A2 also metabolizes a number of procarcinogens and endogenous substrates (Zhou et al., 2009). RA can increase the activity of CYP1A2 in this study. It suggests that RA helps restore hepatic drug metabolism, benefits body discharging exogenous materials and endogenous products and helps to explain the antitoxic mechanism of itself. CYP3A4 is the most abundant CYP found in human liver (40%) metabolizing more than 50% clinically prescribed drugs. Many cancer chemotherapeutic agents such as vinca alkaloids, paclitaxel, irinotecan and other widely used drugs such as amiodarone, diltiazem, lovastatin and midazolam are its substrates (Zhou et al., 2009). The inhibition on CYP3A4 could be highly important for many conditions, which could be of concern, such as the endogenous metabolism, specific physiological functions, the biotransformation of ubiquitous environmental pollutants and changes in the pharmacokinetics of coadministered drugs. With the great use of RA as a herbal medicine, people should pay more attention to its side effects caused by drug–drug interactions when they are administrated with other drugs, especially with substrates of CYP3A4.
Conclusion In conclusion, the inconspicuous effects of RA in vivo on probes of CYP2C9 and CYP2E1 metabolism suggest that there is no clinically relevant drug–drug interaction between the drugs metabolized by these enzymes and RA when
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Table 4. Main pharmacokinetic parameters of midazolam after multiple doses of RA in rat plasma (n ¼ 6, mean SD). Parameter t1/2 (h) Tmax(h) Cmax (ng/mL) AUC(0–1) (mgh/L) MRT(0–1) (h) CL (L/h/kg)
BCG
LDG
MDG
HDG
5.365 0.191 0.247 0.004 6256.714 127.088 39 312.245 1602.686 7.464 0.588 0.254 0.004
5.717 0.250 0.266 0.005 6554.429 152.446 44 292.342 1159.704* 8.024 0.652 0.226 0.006*
5.739 0.168 0.274 0.005 6768.429 149.608 51 103.299 2184.750* 8.246 0.927* 0.196 0.003*
6.728 0.207* 0.298 0.004* 7063.143 182.872* 62 943.242 2433.412** 9.686 0.637* 0.159 0.003**
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*Significantly different from control, p50.05. **Significantly different from control, p50.01.
Figure 4. Mean plasma concentration–time curves of midazolam in rats.
they are used concomitantly. However, from our present results, we cannot exclude that comedication of RA with drugs metabolized by human CYP1A2 and CYP3A4 may induce or inhibit metabolism of these drugs and change plasma concentrations of these drugs, which will result in relevant drug–drug interaction. Further clinical studies are required to fully assess the safety of RA in terms of CYP1A2 induction and CYP3A4 inhibition.
Declaration of interest The authors report no conflicts of interest.
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