http://informahealthcare.com/xen ISSN: 0049-8254 (print), 1366-5928 (electronic) Xenobiotica, Early Online: 1–8 ! 2015 Informa UK Ltd. DOI: 10.3109/00498254.2015.1017753
RESEARCH ARTICLE
Salvianolic acid B as a substrate and weak catechol-O-methyltransferase inhibitor in rats
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Qu Qi, Lijuan Cao, Feiyan Li, Hong Wang, Huiying Liu, Haiping Hao, and Kun Hao State Key Laboratory of Natural Medicines, Jiangsu Province Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, China Abstract
Keywords
1. The aim of this study was to investigate the biotransformation of salvianolic acid B (SAB) by catechol-O-methyltransferase (COMT) and its interaction with levodopa (L-DOPA) methylation in rats. 2. The enzyme kinetics of SAB were studied after incubation with rat COMT. The in vivo SAB and 3-monomethyl-SAB (3-MMS) levels were determined after a single dose of tolcapone with or without SAB administration. For L-DOPA, the effect of SAB inhibition on L-DOPA methylation was studied in vitro. The L-DOPA and 3-O-methyldopa (3-OMD) levels were determined after single and multiple doses of SAB with or without L-DOPA administration. 3. After incubation, we found that SAB was methylated mainly by rat liver and kidney COMT. Tolcapone strongly inhibited the formation of 3-MMS in vitro and in vivo, without any change in the plasma concentration of SAB. Moreover, tolcapone significantly increased the cumulative bile excretion of SAB from 3% to 40% in the rat. SAB inhibited the methylation of L-DOPA with an IC50 value of 2.08 mM in vitro. In vivo, a single intravenous dose of SAB decreased the plasma concentration of 3-OMD, with no obvious effect on the pharmacokinetics of L-DOPA. Multiple doses of SAB given to rats also decreased the plasma concentration of 3-OMD, while SAB increased the plasma concentration of L-DOPA.
Catechol-O-methyltransferase, levodopa, methylation, pharmacokinetics, salvianolic acid B
Introduction Due to its range of pharmacological uses, danshen (Radix Salvia miltiorrhiza) has been one of the most frequently purchased herbs in China for many years. Because danshen is usually prepared and consumed as a decoction, its watersoluble compounds were believed to be the components that are primarily responsible for its pharmacological effects. Salvianolic acid B (SAB), the most abundant water-soluble component of danshen, has been shown to have several pharmacological benefits (Du et al., 2000; Li et al., 2004). However, the pharmacokinetic basis for its pharmacological effects and its safety profile were poorly explored, and its pharmacokinetic interaction with other drugs required a more rigorous evaluation. As a natural product, the drug-like properties of SAB, including its efficacy, safety parameters, and pharmacokinetic behavior, need to be investigated for its development as a drug candidate. Because of the requirement of demonstrating the efficacy and safety of SAB, its drug disposition must be clarified in Address for correspondence: Prof. Haiping Hao and Kun Hao, State Key Laboratory of Natural Medicines, Jiangsu Province Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, China. Tel: +86 25 83271192. E-mail: [email protected]
History Received 7 January 2015 Revised 5 February 2015 Accepted 7 February 2015 Published online 14 April 2015
detail to develop it as a candidate drug. SAB was predominantly eliminated (86%) into the bile as methylated metabolites within 6 h of its intravenous injection into rats (Li et al., 2007). Four methylated metabolites were found, and 3-monomethyl-SAB (3-MMS) was found to be the most important methylated metabolite of SAB (Li et al., 2007; Zhang et al., 2004). These previous results indicated that methylation is the predominant metabolic pathway for SAB, and catechol-Omethyltransferase (COMT) is likely the major enzyme responsible for SAB metabolism. COMT is a ubiquitous enzyme in many species, and it plays an important role in the methylated metabolism of catechol neurotransmitters and xenobiotics (Bonifacio et al., 2002). COMT is widely expressed in most mammalian tissues, with the highest expression in the liver followed by the kidney and gastrointestinal tract (Mannisto & Kaakkola, 1999). The main interest in COMT lies in the possibility of using a COMT inhibitor as an adjunct to levodopa (L-DOPA) therapy of Parkinson’s disease (Mannisto & Kaakkola, 1990). L -DOPA is always used in combination with a carbidopa to prevent its rapid decarboxylation into dopamine in peripheral tissues so that more L-DOPA will be retained in the blood and can then be transported through the blood–brain barrier. Therefore, the concentration of L-DOPA in the blood is an important parameter in Parkinson’s patients. However, when
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the decarboxylation reaction of L-DOPA was inhibited by carbidopa, nearly 90% of L-DOPA was catalyzed by COMT to form 3-O-methyldopa (3-OMD). For this reason, COMT inhibitors have been suggested as a means of preventing the wearing-off phenomenon in Parkinson’s patients by increasing the concentrations of circulating and central L-DOPA (Schapira et al., 2000). Taken together, potential COMT inhibitors with higher efficacy and lower toxicity need to be developed. Previous investigations have found that some tea polyphenols exert potential inhibitory effects on COMT (Nagai et al., 2004). In the present study, we analyzed the enzyme kinetics of SAB in COMT-containing fractions prepared from the liver, kidney, intestine, and lung, where COMT was highly expressed. The influence of tolcapone, a typical COMT inhibitor, on SAB metabolism was also assessed in vitro and in vivo. Furthermore, the inhibitory effect of SAB on the metabolism of L-DOPA was evaluated in vitro and in vivo.
Materials and methods Chemicals SAB was supplied by the Shanghai Institute of Pharmaceutical Industry (Shanghai, China). S-Adenosyl-Lmethionine (SAM), L-DOPA, and chloromycetin (internal standard) were purchased from Sigma-Aldrich (St. Louis, MO). 3-OMD was purchased from Toronto Research Chemicals Inc. (Toronto, Canada). 3-MMS was prepared by the Artificial Synthesis Department of the Medicinal Chemistry Room of China Pharmaceutical University (Nanjing, China). All other chemicals were obtained from Fisher Scientific Co. (Pittsburgh, PA). Animals Male Sprague–Dawley rats weighing 180–220 g were supplied by the Centre of Experimental Animals of China Pharmaceutical University (Nanjing, China). The rats were acclimated for at least 1 week prior to the experiments and were allowed water and standard chow ad libitum. All the animal studies were approved by the Animal Ethics Committee of China Pharmaceutical University. Preparation of COMT fractions from rat tissues Liver, kidney, intestine, and lung COMT enzymes were prepared as described previously (Bonifacio et al., 2002). In brief, tissues from six male Sprague–Dawley rats were homogenized in phosphate buffer (5 mM, pH 7.4) and centrifuged at 15 000g for 20 min. Subsequently, the supernatants were used in COMT activity assays. The protein concentrations were determined by a BCA protein assay with a commercially available kit from Pierce Chemical (Rockford, IL). Methylation of SAB by rat COMT The COMT reaction was performed as described previously, with some modifications (Bonifacio et al., 2002). The protein concentration and incubation time were optimized to ensure the linear formation of 3-MMS. The final COMT
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protein concentration selected from the liver and kidney was 0.2 mg/mL, while 1 mg/mL was used from the intestine and lung. The incubation time for kidney, intestine, and lung COMT was 30 min, while 20 min was used for liver COMT. The formation of 3-MMS was evaluated by incubating COMT with a series of concentrations of SAB (5–1000 mM). After preincubation for 5 min at 37 C, the reaction was started by adding SAB. All the reactions were incubated at 37 C for 30 min, terminated by the addition of 2 M perchloric acid, and centrifuged at 20 000g for 10 min. Finally, the supernatants were analyzed. All reactions were performed in triplicate. The rate of methylation was expressed as picomoles of 3-MMS formed per milligram protein per minute (pmol/mg protein/ min). The Km and Vmax were calculated with the substrate inhibition equation using the Prism 5 software from GraphPad Software Inc. (San Diego, CA). Effect of tolcapone on the methylation of SAB in vitro Liver COMT was used to evaluate the inhibitory effect of tolcapone on the methylation of SAB in vitro. The Km of SAB methylation was approximately 20 mM with the liver COMT, so this concentration was used in the inhibition study. A series of concentrations of tolcapone (0–200 nM) were added to the reaction, and the other reaction conditions were as described previously. The IC50 value was calculated after transforming the concentration of tolcapone to its log concentration. All reactions were performed in triplicate. Effect of tolcapone on the methylation of SAB in vivo Male Sprague–Dawley rats were allocated into the tolcapone group (SAB + tolcapone) or the control group (SAB + vehicle) with six rats in each group. An oral tolcapone dose of 100 mg/kg (Mannisto & Kaakkola, 1999) or vehicle was given 30 min before an intravenous administration of 50 mg/kg SAB (Yang et al., 2008). Blood was collected from the orbital venous sinus at 0.08, 0.17, 0.33, 0.5, 0.75, 1, 2, and 4 h, and the plasma was stored at 20 C until further analysis. Bile was collected at 0–2, 2–4, 4–8, and 8–12 h from another set of twelve rats that received the same treatment. Next, 50 mL of plasma or 100 mL of bile was acidified by 1 M hydrochloric acid and extracted by 2 mL ethyl acetate. After the extraction was evaporated to dryness, the residue was reconstituted in the mobile phase. The pharmacokinetic parameters were estimated by the non-compartmental method using Phoenix WinNonlin version 6.1 software (Pharsight, St. Louis, MO). Effect of SAB on the methylation of L-DOPA in vitro We used liver COMT fractions to evaluate the effect of SAB on the methylation of L-DOPA. The concentrations of L -DOPA used were 0.5, 1, 2, 5, 10, 20, and 50 mM. A series of SAB concentrations (0–10 mM) were added to the reaction. The 3-OMD levels were measured, and the enzyme kinetic parameters were obtained by the Michaelis–Menten equation. Considering the high protein binding ability of SAB, the influence of SAB protein binding on COMT inhibition was determined by adding 2% bovine serum albumin (BSA) to the reaction. All reactions were performed in triplicate.
Biotransformation of salvianolic acid B
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DOI: 10.3109/00498254.2015.1017753
Effect of SAB on the methylation of L-DOPA in vivo
Results
Male Sprague–Dawley rats were allocated into the SAB group (L-DOPA + SAB) or a control group (L-DOPA + vehicle), with six rats in each group. The rats were intravenously administered a single dose of SAB (50 mg/kg) or vehicle 10 min before the administration of L-DOPA and carbidopa (20 and 5 mg/kg, respectively, i.g.). Blood was collected from the orbital venous sinus at 0.08, 0.17, 0.5, 0.75, 1, 2, 4, 6, 8, and 12 h, and the plasma was stored at 20 C until further analysis. To investigate the influence of multiple administrations of SAB on the methylation of L-DOPA, male Sprague–Dawley rats were allocated into the SAB group (L-DOPA + SAB) or the control group (L-DOPA + vehicle), with six rats in each group. The rats were pretreated with SAB (50 mg/kg, i.p.) or vehicle once a day for 8 d. Ten minutes after the last administration, rats were administered L-DOPA and carbidopa (20 and 5 mg/kg, respectively, i.g.). Blood was collected, and the plasma was prepared as described above.
Verification of SAB metabolites
HPLC-MS/MS detection of SAB and 3-MMS The analysis was performed in a Shimadzu HPLC system (Kyoto, Japan) coupled with an AB Sciex 4000 mass spectrometer from Applied Biosystems (Foster City, CA). HPLC separation was performed using a Hypersil C18 column from the Dalian Elite Company (250 4.6 mm, Dalian, China). Linear gradient elution of mobile phase A (methanol) and B (0.5% aqueous formic acid and 1.5 mM ammonium acetate) was used with a range of 15% A and 85% B to 67.5% A and 32.5% B eluted at 8.5 min and recovered to the initial conditions in 0.5 min. The detection was stopped at 15 min, and the mobile phase was delivered at a flow rate of 0.6 mL/min. HPLC-MS/MS was performed in a negative selected ion-monitoring mode. SAB, 3-MMS and chloromycetin were detected at m/z 717.3–519.5, 731.2–533.4 and 321.1–151.9, respectively. The HPLC-MS/MS experiments found a linear concentration range of 0.1–200 mg/mL SAB and 3-MMS with correlation coefficients greater than 0.99. HPLC-fluorescence detection of L-DOPA and 3-OMD A Shimadzu HPLC-2010C system (Shimadzu Corporation, Kyoto, Japan) with a quaternary pump, auto sampler, column oven, and fluorescence detector was used. The separation was performed using a Shimadzu VP-ODS column (150 4.6 mm, Shimadzu Corporation, Kyoto, Japan). The mobile phase consisted of 5% acetonitrile and 95% water (containing 0.4% aqueous phosphoric acid and 50 mM sodium dihydrogen phosphate) for an isocratic elution. The flow rate was 1 mL/min. The excitation wavelength was at 220 nm, and the emission wavelength was at 320 nm. The quantitative ranges for L-DOPA and 3-OMD were 0.05–5 mg/mL and 0.1–10 mg/mL, respectively. Data analysis Results were expressed as the mean ± SD. The statistical significance was determined by Student’s t test with significance set at p50.05.
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In earlier studies, we performed an HPLC-IT/TOF-MS analysis of SAB metabolites after intravenous injection of SAB into rats. Four metabolites were detected in the plasma and bile, and 3-MMS was the dominant metabolite. However, low levels of SAB and its metabolites were excreted into the urine. In vitro, 3-MMS was also the dominant metabolite, while the other three methylated metabolites could not be quantified. Therefore, we focused on the metabolic profiles of 3-MMS in vitro and in vivo. Enzyme kinetics of SAB methylation by rat COMT The enzyme kinetic plots for the formation of 3-MMS are shown in Figure 1. The formation of 3-MMS fits well with a typical model of substrate inhibition. The enzyme kinetic parameters are presented in Table 1. We found that the formation of 3-MMS in the liver showed the highest apparent CLint, followed by the kidney, intestine, and lungs. Thus, we chose to further evaluate the metabolic profile of SAB in the liver. Effect of tolcapone on the methylation of SAB To verify the role of COMT in the metabolism of SAB, the effect of tolcapone on the formation of 3-MMS was assessed. As expected, tolcapone showed a strong inhibitory effect on 3-MMS formation in a concentration-dependent manner, and its IC50 value was only 17.15 nM (Figure 1). To test whether this effect would also occur in vivo, the influence of tolcapone on SAB pharmacokinetics was studied. The plasma drug concentration profiles are presented in Figure 2(A and B), and the pharmacokinetic parameters are summarized in Table 2. For the SAB pharmacokinetics, no obvious difference was observed between the control group and the tolcapone group. However, the AUC of 3-MMS in the tolcapone group was only 10% of that in the control group. We also analyzed the cumulative bile excretion of SAB and 3-MMS. As shown in Figure 2(C and D), the cumulative bile excretion of SAB increased from 3% in the control group to 40% in the tolcapone group, while 3-MMS excretion decreased, suggesting that tolcapone resulted in a significant inhibition of SAB methylation in vivo. The effect of SAB on the methylation of L-DOPA in vitro The enzyme kinetics for the methylation of L-DOPA with liver COMT is shown in Figure 3(A). SAB inhibited the formation of 3-OMD in a concentration-dependent manner (Figure 3B). The estimated IC50 value was 2.08 mM, indicating that SAB is an inhibitor of COMT in vitro. Considering that SAB is highly bound to plasma proteins, we tested the inhibitory effect of SAB on COMT with or without added protein. As shown in Figure 3(B), the addition of 2% bovine serum albumin (BSA) to the reaction strongly compromised the inhibitory effect of SAB on COMT (IC50: from 2.08 to 7.91 mM).
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Figure 1. Enzyme kinetics of SAB in rat liver, kidney, intestine, and lung after incubation with COMT and inhibition kinetics of tolcapone on SAB methylation in the formation of 3-MMS in the presence of rat liver COMT. All reactions were performed in triplicate.
Table 1. The enzyme kinetic parameters of SAB for the formation of 3-MMS in rat COMT. Liver
Kidney
Intestine
Lung
Km (mM) 79.7 ± 4.2 70.0 ± 1.9 16.2 ± 2.5 11.3 ± 0.8 Vmax (nmol/mg/min) 12.8 ± 2.4 7.0 ± 0.8 0.6 ± 0.2 0.2 ± 0.03 CLint (ml/mg/min) 160.5 ± 8.7 99.9 ± 4.8 37.3 ± 1.9 17.5 ± 1.5
The effect of SAB on the methylation of L-DOPA in vivo Because of the inhibitory effect of SAB on COMT in vitro, we performed animal studies to validate the inhibitory effect in vivo. The plasma pharmacokinetics of L-DOPA and 3-OMD after levodopa/carbidopa administration with or without a single intravenous injection of SAB are shown in
Figure 4(A and B). The t1/2 and AUC are presented in Table 3. A single intravenous injection of SAB into rats had little effect on the pharmacokinetics of L-DOPA; however, the plasma drug concentrations of 3-OMD significantly decreased upon SAB treatment. We further asked whether long-term treatment with SAB would result in a significant inhibitory effect of SAB on COMT. Rats were dosed with SAB once a day for 8 consecutive days and then administered L -DOPA and carbidopa 10 min after the last administration of SAB. The pharmacokinetics of L-DOPA and 3-OMD are shown in Figure 4(C and D) and Table 3. SAB treatment led to a statistically significant increase in the plasma concentrations of L-DOPA after 8 consecutive days of SAB administration. For 3-OMD, a more prominent decrease was found with multiple doses of SAB, compared with a single dose.
Biotransformation of salvianolic acid B
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DOI: 10.3109/00498254.2015.1017753
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Figure 2. Plasma drug concentration (A and B) and percentage of cumulative bile excretion (C and D) of SAB and 3-MMS after intravenous administration of 50 mg/kg SAB with oral administration of vehicle (circles) or 100 mg/kg tolcapone (squares). Data were expressed as the mean ± SD (n¼6). **p50.01 versus the control group.
Table 2. The major pharmacokinetic parameters of SAB and 3-MMS in rats after a single administration. SAB Parameters T1/2 (h) AUC(0-Inf) (mg*h/mL) Bile cumulative excretion (%)
3-MMS
Control group (SAB + vehicle)
Tolcapone group (SAB + tolcapone)
Control group (SAB + vehicle)
Tolcapone group (SAB + tolcapone)
1.7 ± 0.6 81.9 ± 32.0 3.0 ± 0.8
1.6 ± 0.4 83.6 ± 27.5 39.3 ± 14.2**
0.8 ± 0.2 17.5 ± 11.5 15.8 ± 2.1
0.4 ± 0.1* 0.9 ± 0.4** 4.2 ± 1.0**
Data are expressed as mean ± SD (n ¼ 6). **p50.01 or *p50.05 versus the control group.
Figure 3. Enzyme kinetics of L-DOPA in the presence of rat liver COMT (A) and inhibition kinetics of SAB on L-DOPA methylation in the formation of 3-OMD (B) in the presence of rat liver COMT. All reactions were performed in triplicate. **p50.01 or *p50.05 versus the control group.
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Figure 4. The effect of a single administration of SAB (50 mg/kg, i.v.) (A and B) or multiple administrations of SAB (50 mg/kg, i.p.) (C and D) on plasma concentrations of L-DOPA and 3-OMD in rats. The circles represent L-DOPA/carbidopa (20 and 5 mg/kg, respectively, i.g.) and the squares represent SAB+ L-DOPA/carbidopa (20 and 5 mg/kg, respectively, i.g.). Data were expressed as the mean ± SD (n¼6). *p50.05 versus the control group.
Table 3. The major pharmacokinetic parameters of L-DOPA and 3-OMD in rats after a single and multiple administrations of SAB. L-DOPA
Parameters Single administration T1/2 (h) AUC(0-Inf) (mg*h/mL) Multiple administration T1/2 (h) AUC(0-Inf) (mg*h/mL)
3-OMD
Control group (L-DOPA/carbidopa + vehicle)
SAB group (L-DOPA/carbidopa + SAB)
Control group (L-DOPA/carbidopa + vehicle)
SAB group (L-DOPA/carbidopa + SAB)
1.5 ± 0.4 4.1 ± 0.7
1.4 ± 0.3 4.8 ± 0.6
11.4 ± 2.9 110.1 ± 19.2
10.2 ± 2.8 66.1 ± 9.3**
1.2 ± 0.2 3.9 ± 0.6
1.1 ± 0.3 5.4 ± 0.5*
10.1 ± 2.5 116.6 ± 17.9
11.7 ± 2.4 55.1 ± 6.0**
Data are expressed as mean ± SD (n ¼ 6). **p50.01 or *p50.05 versus the control group.
Discussion SAB is a major component of danshen, an herb consumed by humans. Elucidating the characteristics of SAB in vitro and in vivo is crucial. The present study helps to clarify the role of COMT in the metabolism of SAB and the weak inhibitory effect of SAB on COMT. COMT is widely distributed among tissues and plays an important role in metabolizing certain endogenous and exogenous substances that are characterized by their catechol structure (Haasio, 2010). To clarify the role of COMT in SAB metabolism, we first performed an enzyme kinetic assay in COMT-containing fractions prepared from the liver, kidney, intestine, and lung that contained high levels of COMT
expression and high levels of exposure to SAB. We found that the liver and kidney had much higher CLint than other tissues. In the present study, we used total COMT-containing fractions (soluble and membrane-bound COMT) from different tissues for the reactions; however, the metabolic capacity of soluble COMT was higher than that of the membranebound COMT that we found in our other studies (data not shown). The results suggested that soluble COMT may be the major contributor to the methylation of SAB. This result was also consistent with the previous finding that soluble COMT is more highly expressed in tissues than membrane-bound COMT. Tolcapone is a broad-spectrum COMT inhibitor that works in both the periphery and the brain. An oral tolcapone
Biotransformation of salvianolic acid B
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DOI: 10.3109/00498254.2015.1017753
dose of 100 mg/kg can cause a nearly 70% inhibition of rat COMT activity (Mannisto & Kaakkola, 1999; Truong, 2009). In our study, a single dose of tolcapone (100 mg/kg) administered to rats dramatically reduced the formation of 3-MMS in plasma by almost 90% compared with control rats. To our surprise, tolcapone had little influence on the plasma pharmacokinetic profiles of SAB. To address this discrepancy, we further analyzed the cumulative bile excretion of SAB and 3-MMS (no SAB and metabolites in urine). The cumulative bile excretion of SAB was significantly increased while that of 3-MMS decreased in tolcapone-treated rats, suggesting that COMT exerts an important role on the methylation of SAB. The scant influence of tolcapone on the plasma pharmacokinetics of SAB may be due to the fact that SAB was rapidly excreted into the bile within 2 h after dosing. The SAB concentration that should have been increased by tolcapone was attenuated due to the increased cumulative bile excretion of SAB. However, it is important to note that the co-administration of SAB with other COMT inhibitors/substrates may lead to an alteration in the efficacy of SAB because the methylated metabolites of SAB also showed pharmacological activities (Cao et al., 2012; Zhang et al., 2004). SAB is a typical COMT substrate both in vitro and in vivo. Therefore, we asked whether SAB could affect the metabolism and pharmacokinetics of other COMT substrates, such as L-DOPA. L -DOPA must always be administered in combination with a dopadecarboxylase inhibitor (Deane et al., 2004; Muller & Russ, 2006). After a peripheral dopadecarboxylase inhibitor was administered together with L-DOPA, most of the L-DOPA could also be metabolized by COMT in the plasma. For this reason, the administration of COMT inhibitors, such as entacapone and tolcapone, has been suggested as a way of enhancing the level of L-DOPA in the plasma. However, side effects appear after entacapone and tolcapone administration. Therefore, it is of considerable interest to develop new COMT inhibitors with improved safety and efficacy (Borges, 2005; Dingemanse, 2000). Some tea catechins and bioflavonoids have been found to have an inhibitory effect on human liver cytosolic COMT (Nagai et al., 2004). In the present study, the concentration of 3-OMD decreased after a single dose of SAB, suggesting that SAB definitely inhibited the methylation of L-DOPA via COMTs. However, the unchanged L-DOPA concentration in the plasma showed that the L-DOPA level was tightly regulated to maintain its concentration within a narrow range (Buck & Ferger, 2010; Kang et al., 2010). To better explain this phenomenon, a clearer understanding of L-DOPA metabolism in vivo may be necessary. The inhibition of 3-OMD may have induced a compensatory metabolic route for L-DOPA. Previous studies with another COMT inhibitor, EGCG, in rats found similar results to the present findings (Kang et al., 2010). The rapid metabolism and excretion of SAB with a half-life of less than 2 h may be another cause for the poor inhibitory effect in vivo. Because the effect of a single dose of SAB on the L-DOPA level in plasma was not satisfactory, we asked whether longterm treatment with SAB would likely improve the L-DOPA level in the plasma. After long-term treatment with SAB, SAB could decrease the formation of 3-OMD and increase the plasma L-DOPA levels. Despite the modest effects,
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the results seemed to be more significant than that observed with a single dose. In summary, we found that SAB is a typical substrate of COMT. Other COMT inhibitors could change the disposition of SAB and 3-MMS. Moreover, the inhibitory effect of SAB on COMT was confirmed in vitro and in vivo, although it exhibited a relatively limited inhibitory effect in vivo. After long-term exposure with SAB, SAB showed a modest inhibitory effect on COMTs. As a potent substrate and weak inhibitor, it is important to note that the co-administration of SAB with other COMT inhibitors/substrates may lead to pharmacokinetic and pharmacodynamic interactions between SAB and other compounds.
Declaration of interest The authors report no conflicts of interest. This study was financially supported by the Foundation for Natural Science Foundation of China (Grant 81302839) and the Natural Science Foundation of Jiangsu Province (Grant BK2012352).
References Bonifacio MJ, Archer M, Rodrigues ML, et al. (2002). Kinetics and crystal structure of catechol-o-methyltransferase complex with cosubstrate and a novel inhibitor with potential therapeutic application. Mol Pharmacol 62:795–805. Borges N. (2005). Tolcapone in Parkinson’s disease: liver toxicity and clinical efficacy. Expert Opin Drug Saf 4:69–73. Buck K, Ferger B. (2010). L-DOPA-induced dyskinesia in Parkinson’s disease: a drug discovery perspective. Drug Discov Today 15:867–75. Cao W, Guo XW, Zheng HZ, et al. (2012). Current progress of research on pharmacologic actions of salvianolic acid B. Chin J Integr Med 18: 316–20. Deane KH, Spieker S, Clarke CE. (2004). Catechol-O-methyltransferase inhibitors for levodopa-induced complications in Parkinson’s disease. Cochrane Database Syst Rev 18:CD004554. Dingemanse J. (2000). Issues important for rational COMT inhibition. Neurology 55:S24–7. Du GH, Qiu Y, Zhang JT. (2000). Salvianolic acid B protects the memory functions against transient cerebral ischemia in mice. J Asian Nat Prod Res 2:145–52. Haasio K. (2010). Toxicology and safety of COMT inhibitors. Int Rev Neurobiol 95:163–89. Kang KS, Wen Y, Yamabe N, et al. (2010). Dual beneficial effects of (-)-epigallocatechin-3-gallate on levodopa methylation and hippocampal neurodegeneration: in vitro and in vivo studies. PLoS One 5:e11951. Li M, Zhao C, Wong RN, et al. (2004). Inhibition of shear-induced platelet aggregation in rat by tetramethylpyrazine and salvianolic acid B. Clin Hemorheol Microcirc 31:97–103. Li X, Yu C, Lu Y, et al. (2007). Pharmacokinetics, tissue distribution, metabolism, and excretion of depside salts from Salvia miltiorrhiza in rats. Drug Metab Dispos 35:234–9. Mannisto PT, Kaakkola S. (1990). Rationale for selective COMT inhibitors as adjuncts in the drug treatment of Parkinson’s disease. Pharmacol Toxicol 66:317–23. Mannisto PT, Kaakkola S. (1999). Catechol-O-methyltransferase (COMT): biochemistry, molecular biology, pharmacology, and clinical efficacy of the new selective COMT inhibitors. Pharmacol Rev 51:593–628. Muller T, Russ H. (2006). Levodopa, motor fluctuations and dyskinesia in Parkinson’s disease. Expert Opin Pharmacother 7:1715–30. Nagai M, Conney AH, Zhu BT. (2004). Strong inhibitory effects of common tea catechins and bioflavonoids on the O-methylation of catechol estrogens catalyzed by human liver cytosolic catecholO-methyltransferase. Drug Metab Dispos 32:497–504. Schapira AH, Obeso JA, Olanow CW. (2000). The place of COMT inhibitors in the armamentarium of drugs for the treatment of Parkinson’s disease. Neurology 55:S65–8.
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Truong DD. (2009). Tolcapone: review of its pharmacology and use as adjunctive therapy in patients with Parkinson’s disease. Clin Interv Aging 4:109–13. Yang XN, Wang YJ, Liu YS, et al. (2008). Pharmacokinetics of salvianolic acids after intravenous injection, with and without Panax
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quinquefolium protopanaxadiol saponins, in rats. J Ethnopharmacol 117:408–14. Zhang Y, Akao T, Nakamura N, et al. (2004). Magnesium lithospermate B is excreted rapidly into rat bile mostly as methylated metabolites, which are potent antioxidants. Drug Metab Dispos 32:752–7.
RESEARCH ARTICLE
Salvianolic acid B as a substrate and weak catechol-O-methyltransferase inhibitor in rats
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Qu Qi, Lijuan Cao, Feiyan Li, Hong Wang, Huiying Liu, Haiping Hao, and Kun Hao State Key Laboratory of Natural Medicines, Jiangsu Province Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, China Abstract
Keywords
1. The aim of this study was to investigate the biotransformation of salvianolic acid B (SAB) by catechol-O-methyltransferase (COMT) and its interaction with levodopa (L-DOPA) methylation in rats. 2. The enzyme kinetics of SAB were studied after incubation with rat COMT. The in vivo SAB and 3-monomethyl-SAB (3-MMS) levels were determined after a single dose of tolcapone with or without SAB administration. For L-DOPA, the effect of SAB inhibition on L-DOPA methylation was studied in vitro. The L-DOPA and 3-O-methyldopa (3-OMD) levels were determined after single and multiple doses of SAB with or without L-DOPA administration. 3. After incubation, we found that SAB was methylated mainly by rat liver and kidney COMT. Tolcapone strongly inhibited the formation of 3-MMS in vitro and in vivo, without any change in the plasma concentration of SAB. Moreover, tolcapone significantly increased the cumulative bile excretion of SAB from 3% to 40% in the rat. SAB inhibited the methylation of L-DOPA with an IC50 value of 2.08 mM in vitro. In vivo, a single intravenous dose of SAB decreased the plasma concentration of 3-OMD, with no obvious effect on the pharmacokinetics of L-DOPA. Multiple doses of SAB given to rats also decreased the plasma concentration of 3-OMD, while SAB increased the plasma concentration of L-DOPA.
Catechol-O-methyltransferase, levodopa, methylation, pharmacokinetics, salvianolic acid B
Introduction Due to its range of pharmacological uses, danshen (Radix Salvia miltiorrhiza) has been one of the most frequently purchased herbs in China for many years. Because danshen is usually prepared and consumed as a decoction, its watersoluble compounds were believed to be the components that are primarily responsible for its pharmacological effects. Salvianolic acid B (SAB), the most abundant water-soluble component of danshen, has been shown to have several pharmacological benefits (Du et al., 2000; Li et al., 2004). However, the pharmacokinetic basis for its pharmacological effects and its safety profile were poorly explored, and its pharmacokinetic interaction with other drugs required a more rigorous evaluation. As a natural product, the drug-like properties of SAB, including its efficacy, safety parameters, and pharmacokinetic behavior, need to be investigated for its development as a drug candidate. Because of the requirement of demonstrating the efficacy and safety of SAB, its drug disposition must be clarified in Address for correspondence: Prof. Haiping Hao and Kun Hao, State Key Laboratory of Natural Medicines, Jiangsu Province Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, China. Tel: +86 25 83271192. E-mail: [email protected]
History Received 7 January 2015 Revised 5 February 2015 Accepted 7 February 2015 Published online 14 April 2015
detail to develop it as a candidate drug. SAB was predominantly eliminated (86%) into the bile as methylated metabolites within 6 h of its intravenous injection into rats (Li et al., 2007). Four methylated metabolites were found, and 3-monomethyl-SAB (3-MMS) was found to be the most important methylated metabolite of SAB (Li et al., 2007; Zhang et al., 2004). These previous results indicated that methylation is the predominant metabolic pathway for SAB, and catechol-Omethyltransferase (COMT) is likely the major enzyme responsible for SAB metabolism. COMT is a ubiquitous enzyme in many species, and it plays an important role in the methylated metabolism of catechol neurotransmitters and xenobiotics (Bonifacio et al., 2002). COMT is widely expressed in most mammalian tissues, with the highest expression in the liver followed by the kidney and gastrointestinal tract (Mannisto & Kaakkola, 1999). The main interest in COMT lies in the possibility of using a COMT inhibitor as an adjunct to levodopa (L-DOPA) therapy of Parkinson’s disease (Mannisto & Kaakkola, 1990). L -DOPA is always used in combination with a carbidopa to prevent its rapid decarboxylation into dopamine in peripheral tissues so that more L-DOPA will be retained in the blood and can then be transported through the blood–brain barrier. Therefore, the concentration of L-DOPA in the blood is an important parameter in Parkinson’s patients. However, when
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the decarboxylation reaction of L-DOPA was inhibited by carbidopa, nearly 90% of L-DOPA was catalyzed by COMT to form 3-O-methyldopa (3-OMD). For this reason, COMT inhibitors have been suggested as a means of preventing the wearing-off phenomenon in Parkinson’s patients by increasing the concentrations of circulating and central L-DOPA (Schapira et al., 2000). Taken together, potential COMT inhibitors with higher efficacy and lower toxicity need to be developed. Previous investigations have found that some tea polyphenols exert potential inhibitory effects on COMT (Nagai et al., 2004). In the present study, we analyzed the enzyme kinetics of SAB in COMT-containing fractions prepared from the liver, kidney, intestine, and lung, where COMT was highly expressed. The influence of tolcapone, a typical COMT inhibitor, on SAB metabolism was also assessed in vitro and in vivo. Furthermore, the inhibitory effect of SAB on the metabolism of L-DOPA was evaluated in vitro and in vivo.
Materials and methods Chemicals SAB was supplied by the Shanghai Institute of Pharmaceutical Industry (Shanghai, China). S-Adenosyl-Lmethionine (SAM), L-DOPA, and chloromycetin (internal standard) were purchased from Sigma-Aldrich (St. Louis, MO). 3-OMD was purchased from Toronto Research Chemicals Inc. (Toronto, Canada). 3-MMS was prepared by the Artificial Synthesis Department of the Medicinal Chemistry Room of China Pharmaceutical University (Nanjing, China). All other chemicals were obtained from Fisher Scientific Co. (Pittsburgh, PA). Animals Male Sprague–Dawley rats weighing 180–220 g were supplied by the Centre of Experimental Animals of China Pharmaceutical University (Nanjing, China). The rats were acclimated for at least 1 week prior to the experiments and were allowed water and standard chow ad libitum. All the animal studies were approved by the Animal Ethics Committee of China Pharmaceutical University. Preparation of COMT fractions from rat tissues Liver, kidney, intestine, and lung COMT enzymes were prepared as described previously (Bonifacio et al., 2002). In brief, tissues from six male Sprague–Dawley rats were homogenized in phosphate buffer (5 mM, pH 7.4) and centrifuged at 15 000g for 20 min. Subsequently, the supernatants were used in COMT activity assays. The protein concentrations were determined by a BCA protein assay with a commercially available kit from Pierce Chemical (Rockford, IL). Methylation of SAB by rat COMT The COMT reaction was performed as described previously, with some modifications (Bonifacio et al., 2002). The protein concentration and incubation time were optimized to ensure the linear formation of 3-MMS. The final COMT
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protein concentration selected from the liver and kidney was 0.2 mg/mL, while 1 mg/mL was used from the intestine and lung. The incubation time for kidney, intestine, and lung COMT was 30 min, while 20 min was used for liver COMT. The formation of 3-MMS was evaluated by incubating COMT with a series of concentrations of SAB (5–1000 mM). After preincubation for 5 min at 37 C, the reaction was started by adding SAB. All the reactions were incubated at 37 C for 30 min, terminated by the addition of 2 M perchloric acid, and centrifuged at 20 000g for 10 min. Finally, the supernatants were analyzed. All reactions were performed in triplicate. The rate of methylation was expressed as picomoles of 3-MMS formed per milligram protein per minute (pmol/mg protein/ min). The Km and Vmax were calculated with the substrate inhibition equation using the Prism 5 software from GraphPad Software Inc. (San Diego, CA). Effect of tolcapone on the methylation of SAB in vitro Liver COMT was used to evaluate the inhibitory effect of tolcapone on the methylation of SAB in vitro. The Km of SAB methylation was approximately 20 mM with the liver COMT, so this concentration was used in the inhibition study. A series of concentrations of tolcapone (0–200 nM) were added to the reaction, and the other reaction conditions were as described previously. The IC50 value was calculated after transforming the concentration of tolcapone to its log concentration. All reactions were performed in triplicate. Effect of tolcapone on the methylation of SAB in vivo Male Sprague–Dawley rats were allocated into the tolcapone group (SAB + tolcapone) or the control group (SAB + vehicle) with six rats in each group. An oral tolcapone dose of 100 mg/kg (Mannisto & Kaakkola, 1999) or vehicle was given 30 min before an intravenous administration of 50 mg/kg SAB (Yang et al., 2008). Blood was collected from the orbital venous sinus at 0.08, 0.17, 0.33, 0.5, 0.75, 1, 2, and 4 h, and the plasma was stored at 20 C until further analysis. Bile was collected at 0–2, 2–4, 4–8, and 8–12 h from another set of twelve rats that received the same treatment. Next, 50 mL of plasma or 100 mL of bile was acidified by 1 M hydrochloric acid and extracted by 2 mL ethyl acetate. After the extraction was evaporated to dryness, the residue was reconstituted in the mobile phase. The pharmacokinetic parameters were estimated by the non-compartmental method using Phoenix WinNonlin version 6.1 software (Pharsight, St. Louis, MO). Effect of SAB on the methylation of L-DOPA in vitro We used liver COMT fractions to evaluate the effect of SAB on the methylation of L-DOPA. The concentrations of L -DOPA used were 0.5, 1, 2, 5, 10, 20, and 50 mM. A series of SAB concentrations (0–10 mM) were added to the reaction. The 3-OMD levels were measured, and the enzyme kinetic parameters were obtained by the Michaelis–Menten equation. Considering the high protein binding ability of SAB, the influence of SAB protein binding on COMT inhibition was determined by adding 2% bovine serum albumin (BSA) to the reaction. All reactions were performed in triplicate.
Biotransformation of salvianolic acid B
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DOI: 10.3109/00498254.2015.1017753
Effect of SAB on the methylation of L-DOPA in vivo
Results
Male Sprague–Dawley rats were allocated into the SAB group (L-DOPA + SAB) or a control group (L-DOPA + vehicle), with six rats in each group. The rats were intravenously administered a single dose of SAB (50 mg/kg) or vehicle 10 min before the administration of L-DOPA and carbidopa (20 and 5 mg/kg, respectively, i.g.). Blood was collected from the orbital venous sinus at 0.08, 0.17, 0.5, 0.75, 1, 2, 4, 6, 8, and 12 h, and the plasma was stored at 20 C until further analysis. To investigate the influence of multiple administrations of SAB on the methylation of L-DOPA, male Sprague–Dawley rats were allocated into the SAB group (L-DOPA + SAB) or the control group (L-DOPA + vehicle), with six rats in each group. The rats were pretreated with SAB (50 mg/kg, i.p.) or vehicle once a day for 8 d. Ten minutes after the last administration, rats were administered L-DOPA and carbidopa (20 and 5 mg/kg, respectively, i.g.). Blood was collected, and the plasma was prepared as described above.
Verification of SAB metabolites
HPLC-MS/MS detection of SAB and 3-MMS The analysis was performed in a Shimadzu HPLC system (Kyoto, Japan) coupled with an AB Sciex 4000 mass spectrometer from Applied Biosystems (Foster City, CA). HPLC separation was performed using a Hypersil C18 column from the Dalian Elite Company (250 4.6 mm, Dalian, China). Linear gradient elution of mobile phase A (methanol) and B (0.5% aqueous formic acid and 1.5 mM ammonium acetate) was used with a range of 15% A and 85% B to 67.5% A and 32.5% B eluted at 8.5 min and recovered to the initial conditions in 0.5 min. The detection was stopped at 15 min, and the mobile phase was delivered at a flow rate of 0.6 mL/min. HPLC-MS/MS was performed in a negative selected ion-monitoring mode. SAB, 3-MMS and chloromycetin were detected at m/z 717.3–519.5, 731.2–533.4 and 321.1–151.9, respectively. The HPLC-MS/MS experiments found a linear concentration range of 0.1–200 mg/mL SAB and 3-MMS with correlation coefficients greater than 0.99. HPLC-fluorescence detection of L-DOPA and 3-OMD A Shimadzu HPLC-2010C system (Shimadzu Corporation, Kyoto, Japan) with a quaternary pump, auto sampler, column oven, and fluorescence detector was used. The separation was performed using a Shimadzu VP-ODS column (150 4.6 mm, Shimadzu Corporation, Kyoto, Japan). The mobile phase consisted of 5% acetonitrile and 95% water (containing 0.4% aqueous phosphoric acid and 50 mM sodium dihydrogen phosphate) for an isocratic elution. The flow rate was 1 mL/min. The excitation wavelength was at 220 nm, and the emission wavelength was at 320 nm. The quantitative ranges for L-DOPA and 3-OMD were 0.05–5 mg/mL and 0.1–10 mg/mL, respectively. Data analysis Results were expressed as the mean ± SD. The statistical significance was determined by Student’s t test with significance set at p50.05.
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In earlier studies, we performed an HPLC-IT/TOF-MS analysis of SAB metabolites after intravenous injection of SAB into rats. Four metabolites were detected in the plasma and bile, and 3-MMS was the dominant metabolite. However, low levels of SAB and its metabolites were excreted into the urine. In vitro, 3-MMS was also the dominant metabolite, while the other three methylated metabolites could not be quantified. Therefore, we focused on the metabolic profiles of 3-MMS in vitro and in vivo. Enzyme kinetics of SAB methylation by rat COMT The enzyme kinetic plots for the formation of 3-MMS are shown in Figure 1. The formation of 3-MMS fits well with a typical model of substrate inhibition. The enzyme kinetic parameters are presented in Table 1. We found that the formation of 3-MMS in the liver showed the highest apparent CLint, followed by the kidney, intestine, and lungs. Thus, we chose to further evaluate the metabolic profile of SAB in the liver. Effect of tolcapone on the methylation of SAB To verify the role of COMT in the metabolism of SAB, the effect of tolcapone on the formation of 3-MMS was assessed. As expected, tolcapone showed a strong inhibitory effect on 3-MMS formation in a concentration-dependent manner, and its IC50 value was only 17.15 nM (Figure 1). To test whether this effect would also occur in vivo, the influence of tolcapone on SAB pharmacokinetics was studied. The plasma drug concentration profiles are presented in Figure 2(A and B), and the pharmacokinetic parameters are summarized in Table 2. For the SAB pharmacokinetics, no obvious difference was observed between the control group and the tolcapone group. However, the AUC of 3-MMS in the tolcapone group was only 10% of that in the control group. We also analyzed the cumulative bile excretion of SAB and 3-MMS. As shown in Figure 2(C and D), the cumulative bile excretion of SAB increased from 3% in the control group to 40% in the tolcapone group, while 3-MMS excretion decreased, suggesting that tolcapone resulted in a significant inhibition of SAB methylation in vivo. The effect of SAB on the methylation of L-DOPA in vitro The enzyme kinetics for the methylation of L-DOPA with liver COMT is shown in Figure 3(A). SAB inhibited the formation of 3-OMD in a concentration-dependent manner (Figure 3B). The estimated IC50 value was 2.08 mM, indicating that SAB is an inhibitor of COMT in vitro. Considering that SAB is highly bound to plasma proteins, we tested the inhibitory effect of SAB on COMT with or without added protein. As shown in Figure 3(B), the addition of 2% bovine serum albumin (BSA) to the reaction strongly compromised the inhibitory effect of SAB on COMT (IC50: from 2.08 to 7.91 mM).
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Figure 1. Enzyme kinetics of SAB in rat liver, kidney, intestine, and lung after incubation with COMT and inhibition kinetics of tolcapone on SAB methylation in the formation of 3-MMS in the presence of rat liver COMT. All reactions were performed in triplicate.
Table 1. The enzyme kinetic parameters of SAB for the formation of 3-MMS in rat COMT. Liver
Kidney
Intestine
Lung
Km (mM) 79.7 ± 4.2 70.0 ± 1.9 16.2 ± 2.5 11.3 ± 0.8 Vmax (nmol/mg/min) 12.8 ± 2.4 7.0 ± 0.8 0.6 ± 0.2 0.2 ± 0.03 CLint (ml/mg/min) 160.5 ± 8.7 99.9 ± 4.8 37.3 ± 1.9 17.5 ± 1.5
The effect of SAB on the methylation of L-DOPA in vivo Because of the inhibitory effect of SAB on COMT in vitro, we performed animal studies to validate the inhibitory effect in vivo. The plasma pharmacokinetics of L-DOPA and 3-OMD after levodopa/carbidopa administration with or without a single intravenous injection of SAB are shown in
Figure 4(A and B). The t1/2 and AUC are presented in Table 3. A single intravenous injection of SAB into rats had little effect on the pharmacokinetics of L-DOPA; however, the plasma drug concentrations of 3-OMD significantly decreased upon SAB treatment. We further asked whether long-term treatment with SAB would result in a significant inhibitory effect of SAB on COMT. Rats were dosed with SAB once a day for 8 consecutive days and then administered L -DOPA and carbidopa 10 min after the last administration of SAB. The pharmacokinetics of L-DOPA and 3-OMD are shown in Figure 4(C and D) and Table 3. SAB treatment led to a statistically significant increase in the plasma concentrations of L-DOPA after 8 consecutive days of SAB administration. For 3-OMD, a more prominent decrease was found with multiple doses of SAB, compared with a single dose.
Biotransformation of salvianolic acid B
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DOI: 10.3109/00498254.2015.1017753
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Figure 2. Plasma drug concentration (A and B) and percentage of cumulative bile excretion (C and D) of SAB and 3-MMS after intravenous administration of 50 mg/kg SAB with oral administration of vehicle (circles) or 100 mg/kg tolcapone (squares). Data were expressed as the mean ± SD (n¼6). **p50.01 versus the control group.
Table 2. The major pharmacokinetic parameters of SAB and 3-MMS in rats after a single administration. SAB Parameters T1/2 (h) AUC(0-Inf) (mg*h/mL) Bile cumulative excretion (%)
3-MMS
Control group (SAB + vehicle)
Tolcapone group (SAB + tolcapone)
Control group (SAB + vehicle)
Tolcapone group (SAB + tolcapone)
1.7 ± 0.6 81.9 ± 32.0 3.0 ± 0.8
1.6 ± 0.4 83.6 ± 27.5 39.3 ± 14.2**
0.8 ± 0.2 17.5 ± 11.5 15.8 ± 2.1
0.4 ± 0.1* 0.9 ± 0.4** 4.2 ± 1.0**
Data are expressed as mean ± SD (n ¼ 6). **p50.01 or *p50.05 versus the control group.
Figure 3. Enzyme kinetics of L-DOPA in the presence of rat liver COMT (A) and inhibition kinetics of SAB on L-DOPA methylation in the formation of 3-OMD (B) in the presence of rat liver COMT. All reactions were performed in triplicate. **p50.01 or *p50.05 versus the control group.
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Figure 4. The effect of a single administration of SAB (50 mg/kg, i.v.) (A and B) or multiple administrations of SAB (50 mg/kg, i.p.) (C and D) on plasma concentrations of L-DOPA and 3-OMD in rats. The circles represent L-DOPA/carbidopa (20 and 5 mg/kg, respectively, i.g.) and the squares represent SAB+ L-DOPA/carbidopa (20 and 5 mg/kg, respectively, i.g.). Data were expressed as the mean ± SD (n¼6). *p50.05 versus the control group.
Table 3. The major pharmacokinetic parameters of L-DOPA and 3-OMD in rats after a single and multiple administrations of SAB. L-DOPA
Parameters Single administration T1/2 (h) AUC(0-Inf) (mg*h/mL) Multiple administration T1/2 (h) AUC(0-Inf) (mg*h/mL)
3-OMD
Control group (L-DOPA/carbidopa + vehicle)
SAB group (L-DOPA/carbidopa + SAB)
Control group (L-DOPA/carbidopa + vehicle)
SAB group (L-DOPA/carbidopa + SAB)
1.5 ± 0.4 4.1 ± 0.7
1.4 ± 0.3 4.8 ± 0.6
11.4 ± 2.9 110.1 ± 19.2
10.2 ± 2.8 66.1 ± 9.3**
1.2 ± 0.2 3.9 ± 0.6
1.1 ± 0.3 5.4 ± 0.5*
10.1 ± 2.5 116.6 ± 17.9
11.7 ± 2.4 55.1 ± 6.0**
Data are expressed as mean ± SD (n ¼ 6). **p50.01 or *p50.05 versus the control group.
Discussion SAB is a major component of danshen, an herb consumed by humans. Elucidating the characteristics of SAB in vitro and in vivo is crucial. The present study helps to clarify the role of COMT in the metabolism of SAB and the weak inhibitory effect of SAB on COMT. COMT is widely distributed among tissues and plays an important role in metabolizing certain endogenous and exogenous substances that are characterized by their catechol structure (Haasio, 2010). To clarify the role of COMT in SAB metabolism, we first performed an enzyme kinetic assay in COMT-containing fractions prepared from the liver, kidney, intestine, and lung that contained high levels of COMT
expression and high levels of exposure to SAB. We found that the liver and kidney had much higher CLint than other tissues. In the present study, we used total COMT-containing fractions (soluble and membrane-bound COMT) from different tissues for the reactions; however, the metabolic capacity of soluble COMT was higher than that of the membranebound COMT that we found in our other studies (data not shown). The results suggested that soluble COMT may be the major contributor to the methylation of SAB. This result was also consistent with the previous finding that soluble COMT is more highly expressed in tissues than membrane-bound COMT. Tolcapone is a broad-spectrum COMT inhibitor that works in both the periphery and the brain. An oral tolcapone
Biotransformation of salvianolic acid B
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DOI: 10.3109/00498254.2015.1017753
dose of 100 mg/kg can cause a nearly 70% inhibition of rat COMT activity (Mannisto & Kaakkola, 1999; Truong, 2009). In our study, a single dose of tolcapone (100 mg/kg) administered to rats dramatically reduced the formation of 3-MMS in plasma by almost 90% compared with control rats. To our surprise, tolcapone had little influence on the plasma pharmacokinetic profiles of SAB. To address this discrepancy, we further analyzed the cumulative bile excretion of SAB and 3-MMS (no SAB and metabolites in urine). The cumulative bile excretion of SAB was significantly increased while that of 3-MMS decreased in tolcapone-treated rats, suggesting that COMT exerts an important role on the methylation of SAB. The scant influence of tolcapone on the plasma pharmacokinetics of SAB may be due to the fact that SAB was rapidly excreted into the bile within 2 h after dosing. The SAB concentration that should have been increased by tolcapone was attenuated due to the increased cumulative bile excretion of SAB. However, it is important to note that the co-administration of SAB with other COMT inhibitors/substrates may lead to an alteration in the efficacy of SAB because the methylated metabolites of SAB also showed pharmacological activities (Cao et al., 2012; Zhang et al., 2004). SAB is a typical COMT substrate both in vitro and in vivo. Therefore, we asked whether SAB could affect the metabolism and pharmacokinetics of other COMT substrates, such as L-DOPA. L -DOPA must always be administered in combination with a dopadecarboxylase inhibitor (Deane et al., 2004; Muller & Russ, 2006). After a peripheral dopadecarboxylase inhibitor was administered together with L-DOPA, most of the L-DOPA could also be metabolized by COMT in the plasma. For this reason, the administration of COMT inhibitors, such as entacapone and tolcapone, has been suggested as a way of enhancing the level of L-DOPA in the plasma. However, side effects appear after entacapone and tolcapone administration. Therefore, it is of considerable interest to develop new COMT inhibitors with improved safety and efficacy (Borges, 2005; Dingemanse, 2000). Some tea catechins and bioflavonoids have been found to have an inhibitory effect on human liver cytosolic COMT (Nagai et al., 2004). In the present study, the concentration of 3-OMD decreased after a single dose of SAB, suggesting that SAB definitely inhibited the methylation of L-DOPA via COMTs. However, the unchanged L-DOPA concentration in the plasma showed that the L-DOPA level was tightly regulated to maintain its concentration within a narrow range (Buck & Ferger, 2010; Kang et al., 2010). To better explain this phenomenon, a clearer understanding of L-DOPA metabolism in vivo may be necessary. The inhibition of 3-OMD may have induced a compensatory metabolic route for L-DOPA. Previous studies with another COMT inhibitor, EGCG, in rats found similar results to the present findings (Kang et al., 2010). The rapid metabolism and excretion of SAB with a half-life of less than 2 h may be another cause for the poor inhibitory effect in vivo. Because the effect of a single dose of SAB on the L-DOPA level in plasma was not satisfactory, we asked whether longterm treatment with SAB would likely improve the L-DOPA level in the plasma. After long-term treatment with SAB, SAB could decrease the formation of 3-OMD and increase the plasma L-DOPA levels. Despite the modest effects,
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the results seemed to be more significant than that observed with a single dose. In summary, we found that SAB is a typical substrate of COMT. Other COMT inhibitors could change the disposition of SAB and 3-MMS. Moreover, the inhibitory effect of SAB on COMT was confirmed in vitro and in vivo, although it exhibited a relatively limited inhibitory effect in vivo. After long-term exposure with SAB, SAB showed a modest inhibitory effect on COMTs. As a potent substrate and weak inhibitor, it is important to note that the co-administration of SAB with other COMT inhibitors/substrates may lead to pharmacokinetic and pharmacodynamic interactions between SAB and other compounds.
Declaration of interest The authors report no conflicts of interest. This study was financially supported by the Foundation for Natural Science Foundation of China (Grant 81302839) and the Natural Science Foundation of Jiangsu Province (Grant BK2012352).
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