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Extract from the plant Mitragyna speciosa has been widely used as an opium substitute and as a traditional medicine, mainly due to its morphine-like pharmacological effects. This study investigated the effects of Mitragyna speciosa alkaloid extract (MSE) on human recombinant cytochrome P450 (CYP450) enzyme activities in vitro by using a modified Crespi method. When compared to the more commonly-used LC-MS/MS method, this method has the advantage of being a fast and cost effective way to perform CYP inhibition studies. The results showed that MSE showed a most potent inhibitory activity against CYP3A4 and CYP2D6 with an apparent IC50 value of 0.78 Âµg/ml and 0.636 Âµg/ml. The MSE was moderately potent in inhibiting CYP1A2 with an IC50 of 39 Âµg/ml. The IC50 of CYP2C19 could not be determined due to inhibition of less than 50%. The MSE showed low to moderate CYP inhibition as compared with the well-established positive controls of the various CYP enzymes, namely, quinidine (CYP2D6), ketoconazole (CYP3A4), tranylcypromine (CYP2C19) and furafyllin (CYP1A2). Competitive inhibition was observed in MSE-treated CYP2D6 inhibition assay, while mixed type inhibition was observed in inhibition assay using CYP3A4, 1A2 and 2C19. The results of the study showed that MSE may contribute to herb-drug interaction if administered concomitantly with drugs which are substrates for CYP3A4, CYP2D6 and CYP1A2.
Mitragyna speciosa Kroth is a tropical herb plant which is found mainly in southeast Asian countries such as Thailand and Malaysia. It is also known as "Biak-biak" or "Ketum" in Malaysia, and as "Kratom" in Thailand (Saidin et al., 2008). This plant belongs to the family Rubiaeceae. The major alkaloid present in the leaves of this plant is mitragynine (Juzaili et al., 2010). The leaves of this plant have been traditionally consumed by the Thai and Malaysian natives by chewing the fresh leaves, smoking or drinking as tea, mainly for its stimulant and euphoric effect (Kavita et al., 2008). The leaves have been reported to possess morphine-like properties such as antitussive, anaesthetic, antinociceptive, analgesic and stimulative effects in human and rodents (Suchitra et al., 1998). Due to its opioid-like effects, Kratom has been widely used to treat pain and as an opium substitute in opium withdrawal since the nineteenth century. However, addiction and several opioid abstinence syndrome such as irritability, yawning, rhinorrhoea, myalgias, diarrhoea, tremor, nausea, nystagmus and arthralgia have also been reported (Kavita et al., 2008) thereby limiting its use.
Although the pharmacological effects of Kratom in human and animals have been well established, the doses required to produce stimulation, analgesia and toxicity in humans remain poorly defined. Abuse of the plant by drug addicts when used as an opium substitute has caused major concern in Malaysia and Thailand. Consequently, the kratom plant has been listed as a controlled substance in Malaysia, Thailand and Austrialia. However, in other parts of the world, kratom is currently not strict regulated. The access to kratom through the internet links has caused significant drug abuse among the western population who might use for self-treatment in opioid withdrawal and to get relief in chronic pain (Kavita et al., 2008; Somsmorn et al., 2008).
Cytochrome P450 (CYP) is a major family of enzymes that are involved in the metabolism of drugs, toxicants and endogenous compounds. Metabolism is generally regarded as a protective mechanism by resulting in the production of inert metabolites, but it can also cause toxicity by activation of pro-drugs to active metabolites. Members of the CYP subfamilies exhibit relatively strict specificities in the metabolism of xenobiotics. The CYP1, CYP2 and CYP3 subfamilies are responsible for the metabolism of more than 90% of commercially available drugs (Porrogi et al., 2008). Early knowledge about the metabolism of a new chemical entity and its affinity to certain metabolising enzymes will help in drug development while avoiding any undesirable drug-drug interaction which may lead to changes in the rate of drug metabolism thereby potentially contributing to drug toxicity (Hanapi et al., 2010). For example, ketoconazole and quinidine are well known CYP 3A4 inhibitors which may induce life-threatening heart rhythm disorders when co-administered with other substrate of CYP3A4 such as erythromycin (Ito et al., 2003; Chris et al., 2000).
In order to avoid pharmacokinetic drug-drug interaction due to the CYP inhibition, there exist some methods to measure enzyme activity and enzyme inhibition which can be categorized according to their experimental system and screening strategies (Stephen et al., 2008). In the the process of drug development, fluorescene measurement method has the advantage of being less time consuming and more cost effective. In this method, pro-fluorescence substrate is broken down to a fluorescent product by CYP enzymes and detected directly using a fluorescense plate reader. With the development of sensitive liquid chromatography-tamdem mass spectometry (LC-MS)/MS analytical instrument, the analysis of metabolite generation from an unlabelled drug substrates has become more efficienct and highly specific. However, drug-drug interaction asssessment using LC-MS/MS is considered as moderate throughput and significant investment in analytical equipment is required. In 2006, Food and Drug Administration (FDA) had drafted a guideline for industry on drug interaction studies to reflect the importance of drug metabolism study as a part of new drug safety and effectiveness assessment.
The objective of this study was to evaluate the effect of Mitragyna speciosa alkaloid extract (MSE) on cytochrome P450 enzymes by using a novel high throughput in vitro fluorescent P450 assays. The Mitragyna speciosa extract was tested for its effect on CYP3A4, CYP2D6, CYP1A2 and CYP2C19 to determine its risk potential in causing possible interactions with other therapeutic products.
MATERIALS AND METHODS
Chemicals: Recombinant human cytochrome P450 3A4, 2D6, 1A2, and 2C19 enzymes; marker substrates 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-mthoxy-4-methylcoumarin (AMMC), 7-Benzyloxy-4-(trifluoromethyl)-coumarin (BFC) and 7-Hydroxy-4-(trifluromethyl)-coumarin (HFC); NADPH regeneration system were purchased from GENTEST Corp, while 3-Cyano-7-ethoxycoumarin (CEC) was obtained from Sigma Aldrich, USA. All the other chemicals and standard references including quinidine, ketoconazole, tranylcypromine, and furafyllin were purchased from Sigma Aldrich.
Plant Extraction: Fresh leaves of Mitragyna speciosa Korth were collected from the forest in the state of Perlis in Malaysia. A methanol:chloroform extraction method was used to extract the alkaloid compounds. The leaves (5kg) were dried and soaked in methanol for 3 days. The methanol was filtered and the filtrate was evaporated using a rotary evaporator. The extraction and evaporation procedure was repeated three times. After that, the crude methanol extract was re-dissolved in 10% acetic acid and then washed with hexane. The acidic layer was basified to pH 9 using ammonia hydroxide and extracted with chloroform. The collected organic layer was filtered through anhydrous sodium sulphate and the filtrate was extracted using rotary evaporator to obtain 5g of crude alkaloid extract. The alkaloid extract was then dissolved in DMSO and the presence of alkaloid was confirmed using the Dragendorf test (Juzaili et al., 2010).
Flourometric Enzyme Inhibition Assays. Time and Concentration Linearity. The fluorescence readings of the corresponding metabolites were measured at 10, 20, 30, 45, 60, 90 minute after adding the NRS solution. Km values which had been earlier been established by Crespi et al., (1997) were used as the substrate concentration in this assay. Optimal incubation times were calculated based on the linearity of the graph.
Determination of Km and Vmax values. Reactions were carried out with 10 different substrate concentrations: BFC (1-200 ÂµM), AMMC (0.04-100 ÂµM), CEC (0.13-100 ÂµM) for CYP1A2, and CEC (0.23-300 ÂµM) for CYP2C19 in three fold dilutions. The previously determined optimal incubation times were used thoughout the experiment. Km and Vmax values for each of the CYP enzyme was determined using Michealis-Menten plots. The concentration of DMSO should not exceed more than 0.2% and the concentration of ACN was not more than 2%. DMSO and ACN have been known to be potent inhibitors of CYP enzymes.
IC50 determination of inhibitors and plant extracts. The assays were carried out using the modified Crespi method (Crespi et al., 1997). Incubations were conducted in a total reaction volume of 150Âµl in 96-well fluoroluxTM HB Black, flat bottom microplates from DYNEX, Germany. The final concentration of the plant extracts (0.05 -1000 Âµg/ml) and positive controls (0.005-100ÂµM) were prepared in 3X diution method. NADPH regeneration system (NRS) was prepared using 3.3mM glucose-6-phosphate (G6P), 0.06 U of glucose-6-phosphate dehydrogenase (G6PDH), 3.3mM of MgCl2, and 1.3mM of nicotinamide adenine dinucleotide phosphate (NADP+). The reaction was initiated by addition of 20 Âµl of NRS into a mixture of 30 Âµl pre-warmed enzyme/substrate (E/S) mix, 20 Âµl test compound and 80 Âµl of buffer, followed by 30 minute incubation at 37oC. Following that, the reactions were terminated by addition of 75 Âµl Stop solution (4:1 acetonitrile: 0.5M Tris base). The released fluoroscence was scanned using a fluorescence plate scanner, VICTORTM X5, Perkin Elmer, at the respective optimal wavelength of the metabolite. Positive and negative controls were run with every assay. Data were exported and analyzed using the PRISM software. The IC50 was calculated using relative IC50 determination. The results were presented as mean of three replicates for at least two independent experiments. The protocol was summarized in the Table 1.
Determination of Ki values and modes of inhibition. Apparent inhibition constant (Ki) values were further determined for concentrations of substrates ranging from 3.125-100 ÂµM at different concentrations of MSE (0.125-300 Âµg/ml). The Lineweaver-Burk plots, Dixon plots and secondary reciprocal plots were plotted to determine the Ki values and mode of inhibition.
Determiniation of optimal incubation time. The calibration curves were prepared at different incubation times. Optimal incubation times were determined when good linearity were obtained in which r2 value approached 1.0. At substrate concentration of 50 ÂµM BFC, CYP 3A4 dependent metabolism was linear to 30 minutes with enzyme concentration up to 1.0pmol/well. For CYP1A2 inhibition assay, the optimal incubation time was 20 minutes when CYP1A2 concentration was 0.5pmole/well. The CYP2C19 dependent formation of CEC metabolite was linear to 30 minutes with enzyme concentration up to 0.5pmole/well. For CYP2D6-dependent formation, AMMC metabolite formation increased with time and was optimum at 30 mins incubation time. For all the CYP inhibition assays, the formation of fluoroscence metabolites is proportional with the substrate concentration and with incubation time. In order to determine the Km and Vmax values, the optimal incubation time obtained from the previous experiment was chosen.
Determination of Km and Vmax values. The assay was designed according to Crespi method et al. (1997) in that Km and Vmax values were determined using ten substrate concentration generated by 1:3 dilutions (1-300ÂµM). However, several aspects were modified to minimize the cost and the differences among the assays such as nonspecific binding of compound to the microsome. At concentration above 300ÂµM, a precipitation was observed. The Km values for CYP3A4, CYP2D6, CYP1A2 and CYP2C19 were 48.94, 1.016, 23.69, 4.627ÂµM respectively. The Km values estimated in this study were similar to values quoted in the Crespi et al., 1997. The Vmax values for CYP3A4, CYP2D6, CYP1A2 and CYP2C19 were found to be 2343, 47.18, 20307, 2087 rfu product/min/pmole P450 respectively. The velocity of the reaction showed a hyperbolic pattern and approached the maximum velocity with increasing substrate concentration. The Km values of the standard inhibitor are summarized in the Table 2 and Michaelis-Menten Plots for P450 3A4, 2D6, 1A2 and 2C19 are shown in Fig.1. In order to detect competitive inhibition with comparable efficiency, substrate concentration used for the subsequent assay was close to the apparent Km values.
Determination of IC50 for standard inhibitors. The IC50 values for each of the P450 enzymes were determined using a single concentration of enzyme and substrate which had been optimized previously. The standard inhibitor IC50 values were then compared with the literatures and with the data bank in Aurigene Discovery Technologies (Table 2). IC50 values determinations were performed with quinidine (CYP2D6), ketoconazole (CYP3A4), tranylcypromine(CYP2C19), and furafyllin (CYP1A2) as positive controls. It has been demonstrated that quinidine, ketoconazole, tranylcypromine, and furafyllin are both specific and potent inhibitors of specific liver CYP enzymes (Crespi et al., 1997; Pelkonen et al., 1998; FDA guidance for industry, 1997).
Determination of IC50 for MSE. Standard positive inhibitors and MSE were added separately to the in vitro drug metabolizing systems at varying concentrations (2-10ÂµM for standard positive inhibitors and 0.05-100Âµg/ml for MSE). The CYP-dependant inhibition was observed only at doses lower than 250Âµg/ml MSE. High concentrations of MSE was found to interfere with the fluoroscene measurement (Saidin et al., 2008). Our finding showed that MSE was a strong inhibitor of CYP 3A4 and 2D6 with IC50 of 0.78 Âµg/ml and 0.636 Âµg/ml. MSE appear to have a moderate inhibition of CYP1A2 with IC50 of 39 Âµg/ml. However, the IC50 of CYP2C19 was not determined due to inhibition of less than 50% (Table 3 and Fig 1). The IC50 values for the standard positive inhibitors in this study were very close to the reference values, indicating that the condition of the in vitro system was suitable for subsequent pharmacokinetic studies (Donato et al., 2004).
Determination of Ki values and modes of inhibition for MSE. The most important measurement for a compound as an indication of its inhibitory potency is the Ki, which is the inhibition constant. It is an indication of the affinity of a compound for an enzyme. Various concentrations of substrates were incubated with the respective CYP isoforms in the absence and presence of different concentrations of MSE. The inhibition constants (Ki) and modes of inhibition for MSE in different CYP activities were summarized in Table 3. MSE showed a mixed inhibition on CYP 3A4, 1A2 and 2C19 in which the apparent Km was increased together with a decrease in Vmax. The reduction of the "active" enzyme results in a decrease of Vmax. However, MSE was a competitve inhibitor for CYP2D6 as evident from increasing values of Km with Vmax remaining unchanged (Fig 2).
Since the 1980s, studies on specific forms of CYP450 using in vivo and in vitro systems have attracted the attention of researchers due to its most interesting characteristic of substrate specificity and inhibitor selectivity. Currently, more than 50% of the drugs in the market are metabolized by CYP450 enzymes (Hanapi et al., 2010). The most important CYP450 enzyme is CYP3A4 which is involved in the liver metabolism of >50% of drugs (Akansha et al., 2008). CYP2D6 is found to be relatively specific on positively charged molecules with a basic nitrogen. CYP1A2 on the other hand, is involved in the metabolism of poly-aromatic hydrocarbons and CYP2C9 metabolizes weakly anionic molecules (Akansha et al., 2008). Most organic compounds are therefore able to be metabolized by CYP enzymes. It should be kept in mind that the kinetic characteristic of enzymes are important for metablism and clearance of drugs (Pelkonen et al., 1998). In this study, CYP1A2 is relatively high in reaction velocity as compared to other CYPs, and whilst CYP2D6 showed the lowest reaction velocity; CYP3A4 and CYP2C19 were reported to have moderate rate of reaction. There was a relatively large difference between the Vmax values in this study from reported values in the literature (Crespi et al., 1997; Pelkonen et al., 1998). This may be due to the different sources of the human liver microscomes such as P450-expression cell lines and baculovirus infected insect cells (BTI-TN-5B1-4) (Donato et al., 2004; Stephen et al., 2008). Differences between the functional level of expression system such as protein, lipid, co-enzyme concentration, ratios of reductase and cytochrome b5, phospholipid composition of microsomes and incubation conditions adopted in different laboratories may bring about the significant changes in the Vmax and Km values (Stephen et al., 2008).
Mitragynine has been reported to be the most abundant alkaloid in the extracts from Mitrgyna Speciosa (Reanmongkol et al., 2007). It has been reported to have some morphine-like properties and side effects such as analgesia, respiratory depression and cough suppression (Ruan et al., 2007). It is interesting to note that mitragynine is structurally different from morphine and yet the two produce similar pharmacological effects. Other active constituents such as speciofoline, rhychophylline and stipulatine found in the extract are suspected to contribute to the inhibition to some extent (Beckett et al. 1964). However, the active substances of Mitragyna speciosa that is responsible for substrate metabolism in this study still remain unknown and thus underestimation of the total inhibitory potential of the compound may occur. However, the study using the alkaloid extract may provide the fundamental and representative data on the isoforms examined since usually, people orally consume the herb as a whole leaf instead of consuming a particular active ingredient.. When comparing our results of the IC50 of MSE with that of Hanapi et al., 2010, our study showed a similar pattern of inhibition but with a stronger inhibitory effect. There appears to be a 100-fold stronger inhibition for CYP3A4 and a 6-fold inhibition for CYP2D6 when compared to the results of our study. This may due to the variability and difficulties when working with natural products. Unlike conventional single active compounds, natural products are complex mixtures which contain various chemical substrates that can be variable due to the environmental factors such as climate, growth conditions, harvest and storage conditions (Akansha et al., 2008; Hanapi et al., 2010). In addition, variations such as manner of preparation and extraction methods can also contribute to the data variability. Meanwhile, very weak inhibition of CYP2C9 was reported by Hanapi et al., 2010.
In this study, MSE was found to be a mixed-competitive inhibitor of CYP3A4, 1A2 and 2C19 in vitro with Ki values of 1.526, 18.57 and 84.88 Âµg/ml, respectively. Mixed inhibition is a combination of competitive and uncompetitive enzyme inhibition in which the inhibitor binds to the side other than to the active site (allosteric site) of the free enzyme (E) or of the enzyme substrate (ES) complex. The presence of the inhibitor cause a change in the stucture or shape of the enzyme and is accompanied by decreasing affinity of the enzyme for the substrate. Increased Km value and decreased maximum enzyme reaction rate Vmax was observed from the graph in Fig 2. However, Dixon plot (1/v against i (inhibitor)) alone does not always distinguish between uncompetitve and mixed competitive inhibiton (Athel et al., 1974). Thus, graph s/v against i were plotted in order to provide a good estimation of mode of inhibiton (Fig 3). Graphs 3a, 3b and 3c showed the mixed competition in which the intersection in the Dixon plot was given by i = -Ki (Athel et al., 1974). The intersection that was found in the Dixon plot (1/v against i) also showed that MSE was a mixed inhibitor for CYP 3A4, 1A2 and 2C19 (not shown). On the other hand, MSE was a competitive inhibitor for CYP2D6 with a Ki value of 2.6 Âµg/ml. In competitve inhibition, the substrate competes with the inhibitor for binding to the active site of the enzyme . Therefore, the inhibition can be overcome at sufficiently high substrate concentrations with the VmaxÂ remaining unaffected. Besides, the condition Ki = âˆž (the lines are parallel) was found in the Dixon plot (s/v against i) whereas there is an intersection was found in the conventional Dixon plot 1/v against i (Athel et al., 1974). No report presently exist regarding the effect of MSE on mode of inhibition of CYP enzyme, hence no comparison could be made with the present data.
Co-administration of drugs and herbs which are metabolised by the CYP enzymes mentioned above will have the potential to cause the abolition of metabolic clearancein vivo. This can result in unwanted toxic effects. Hence, the study of herbs as potential drug inhibitors is important to minimize the unwanted consequences of herb-drug interactions. The potential of the Mitragyna speciosa extract to affect the drug clearance and deposition may increase if used in combination with one or more drugs which are substrates for CYP3A4, 2D6 and 1A2 and also with other herbs which are known substrates of CYP3A4 and other isoforms of these CYP enzymes such as St. John's wort, garlic, gingko, ginseng and grapefruit which have been reported to have CYP inhibition properties (Henderson et al., 2002; Zou et al., 2002). Hence these can potentially enhance or inhibit the effects of Mitragyna speciosa (Akansha et al., 2008). High dose of Mitragyna speciosa extract might taken by the chronic users because of its tolerance effects. Since MSE showed a potent inhibitory effect on CYP 3A4 and 2D6 which are the two meatbolising enzymes which are involved in the metabolism of the majority of drugs, with IC50 and Ki values lower than 20 Âµg/ml (Pan et al., 2009; Zou et al., 2002), it is therefore necessary for the patients and doctors to be aware of potential drug interaction when co-administered with other medications.
In conclusion, the modified Crespi CYP inhibition method that is used in this study is a relatively cost effective and faster way to perform CYP inhibition assay. In this study, Mitragyna speciosa alkaloid extract inhibits CYP enzymes with varying degrees of potency. Based on the Ki and IC50 values, the results showed that MSE is a potent inhibitor of CYP3A4 and CYP2D6 (Ki and IC50 values â‰¤ 20 Âµg/ml) implying a high potential risk of herb-drug interactions especially when patients are consuming large amounts of the extracts. The extract was found to moderately inhibit CYP1A2 (IC50 values 20-100Âµg/ml) with no significant inhibition of CYP2C19 (<50% inhibition at highest concentration of MSE). Competitive inhibition was observed for CYP2D6 and mixed inhibition was found for the other CYP isoforms. At present, the active constituent responsible for the CYPs inhibiton still remain unknown and further work is required to identify the inhibition property of each of the active constituents of the Mitragyna speciosa extracts.
This research was supported in part by Aurigene Discovery Technologies (M) Sdn. Bhd and the University of Malaya Grant. The work of my colleagues who assisted me in the projects is gratefully acknowledged.
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Table 1: Summary of the components of the flourometric enzyme inhibition assays.
NADP+ : 1.3mM
NADP+ : 8.3ÂµM
G6P : 3.3mM
G6P : 0.41ÂµM
MgCl2 : 3.3mM
MgCl2 : 0.41ÂµM
G6PDH : 0.06 U/well
G6PDH : 0.06 U/well
Ex: 409 nm
Ex: 409 nm
Ex: 409 nm
Ex: 390 nm
Em: 530 nm
Em: 460 nm
Em: 460 nm
Em: 460 nm
Table 2: Km and IC50 values of the standard inhibitors reported in literature. 7-Benzyloxy-4-(trifluoromethyl)-coumarin (BFC), 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-mthoxy-4-methylcoumarin (AMMC), 3-Cyano-7-ethoxycoumarin (CEC).