Publications by authors named "Chin Eng Ong"

28 Publications

  • Page 1 of 1

inhibitory effects of glucosamine, chondroitin and diacerein on human hepatic CYP2D6.

Drug Metab Pers Ther 2021 Apr 8. Epub 2021 Apr 8.

School of Pharmacy, International Medical University, No. 126, Jalan Jalil Perkasa 19, Bukit Jalil, 57000Kuala Lumpur, Malaysia.

Objectives: Glucosamine, chondroitin and diacerein are natural compounds commonly used in treating osteoarthritis. Their concomitant intake may trigger drug-natural product interactions. Cytochrome P450 (CYP) has been implicated in such interactions. Cytochrome P450 2D6 (CYP2D6) is a major hepatic CYP involved in metabolism of 25% of the clinical drugs. This study aimed to investigate the inhibitory effect of these antiarthritic compounds on CYP2D6.

Methods: CYP2D6 was heterologously expressed in . CYP2D6-antiarthritic compound interactions were studied using enzyme kinetics assay and molecular docking.

Results: The high-performance liquid chromatography (HPLC)-based dextromethorphan -demethylase assay was established as CYP2D6 marker. All glucosamines and chondroitins weakly inhibited CYP2D6 (IC values >300 µM). Diacerein exhibited moderate inhibition with IC and values of 34.99 and 38.27 µM, respectively. Its major metabolite, rhein displayed stronger inhibition potencies (IC=26.22 μM and =32.27 μM). Both compounds exhibited mixed-mode of inhibition. molecular dockings further supported data from the study. From - extrapolation, rhein presented an area under the plasma concentration-time curve (AUC) ratio of 1.5, indicating low potential to cause inhibition.

Conclusions: Glucosamine, chondroitin and diacerein unlikely cause clinical interaction with the drug substrates of CYP2D6. Rhein, exhibits only low potential to cause inhibition.
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http://dx.doi.org/10.1515/dmdi-2020-0182DOI Listing
April 2021

The CYP2R1 Enzyme: Structure, Function, Enzymatic Properties and Genetic Polymorphism.

J Pharm Pharm Sci 2021 ;24:94-112

Monash University Malaysia.

Since the discovery of its role in vitamin D metabolism, significant progress has been made in the understanding of gene organisation, protein structure, catalytic function, and genetic polymorphism of cytochrome P450 2R1 (CYP2R1). Located on chromosome 11p15.2, CYP2R1 possesses five exons, unlike most other CYP isoforms that carry nine exons. CYP2R1 crystal structure displays a fold pattern typical of a CYP protein, with 12 a-helices as its structural core, and b-sheets mostly arranged on one side, and the heme buried in the interior part of the protein. Overall, CYP2R1 structure adopts a closed conformation with the B' helix serving as a gate covering the substrate access channel, with the substrate vitamin D3 occupying a position with the side chain pointing toward the heme group. In liver, CYP2R1 25-hydroxylates vitamin D and serves as an important determinant of 25(OH)D level in the tissue and in circulation. While substrate profile has been well studied, inhibitor specificity for CYP2R1 requires further investigation. Both exonic and non-exonic single nucleotide polymorphisms (SNPs) have been reported in CYP2R1, including the CYP2R1*2 carrying Leu99Pro exchange, and a number of non-exonic SNPs with variable functional consequences in gene regulation. A non-exonic SNP, rs10741657, has its causal relationship with diseases established, including that of rickets, ovarian cancer, and multiple sclerosis. The role of other CYP2R1 SNPs in vitamin D deficiency and their causal link to other traits however remain uncertain currently and more studies are warranted to help identify possible physiological mechanisms underlying those complex traits.
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http://dx.doi.org/10.18433/jpps31305DOI Listing
January 2021

The Molecular and Enzyme Kinetic Basis for Altered Activity of Three Cytochrome P450 2C19 Variants Found in the Chinese Population.

Curr Mol Pharmacol 2020 ;13(3):233-244

School of Pharmacy, International Medical University, 126, Jalan Jalil Perkasa 19, Bukit Jalil, 57000 Kuala Lumpur, Malaysia.

Background: There is a large inter-individual variation in cytochrome P450 2C19 (CYP2C19) activity. The variability can be caused by the genetic polymorphism of CYP2C19 gene. This study aimed to investigate the molecular and kinetics basis for activity changes in three alleles including CYP2C19*23, CYP2C19*24 and CYP2C19*25found in the Chinese population.

Methods: The three variants expressed by bacteria were investigated using substrate (omeprazole and 3- cyano-7-ethoxycoumarin[CEC]) and inhibitor (ketoconazole, fluoxetine, sertraline and loratadine) probes in enzyme assays along with molecular docking.

Results: All alleles exhibited very low enzyme activity and affinity towards omeprazole and CEC (6.1% or less in intrinsic clearance). The inhibition studies with the four inhibitors, however, suggested that mutations in different variants have a tendency to cause enhanced binding (reduced IC50 values). The enhanced binding could partially be explained by the lower polar solvent accessible surface area of the inhibitors relative to the substrates. Molecular docking indicated that G91R, R335Q and F448L, the unique mutations in the alleles, have caused slight alteration in the substrate access channel morphology and a more compact active site cavity hence affecting ligand access and binding. It is likely that these structural alterations in CYP2C19 proteins have caused ligand-specific alteration in catalytic and inhibitory specificities as observed in the in vitro assays.

Conclusion: This study indicates that CYP2C19 variant selectivity for ligands was not solely governed by mutation-induced modifications in the active site architecture, but the intrinsic properties of the probe compounds also played a vital role.
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http://dx.doi.org/10.2174/1874467212666191111110429DOI Listing
January 2020

Functional and structural characterisation of common cytochrome P450 2D6 allelic variants-roles of Pro34 and Thr107 in catalysis and inhibition.

Naunyn Schmiedebergs Arch Pharmacol 2019 08 26;392(8):1015-1029. Epub 2019 Apr 26.

School of Pharmacy, International Medical University, 126, Jalan Jalil Perkasa 19, Bukit Jalil, 57000, Kuala Lumpur, Malaysia.

One major source of inter-individual variability in drug pharmacokinetics is genetic polymorphism of the cytochrome P450 (CYP) genes. This study aimed to elucidate the enzyme kinetic and molecular basis for altered activity in three major alleles of CYP2D6, namely CYP2D6*2, CYP2D6*10 and CYP2D6*17. The E. coli-expressed allelic variants were examined using substrate (venlafaxine and 3-cyano-7-ethoxycoumarin[CEC]) and inhibitor (quinidine, fluoxetine, paroxetine, terbinafine) probes in enzyme assays as well as molecular docking. The kinetics data indicated that R296C and S486T mutations in CYP2D6*2 have caused enhanced ligand binding (enhanced intrinsic clearance for venlafaxine and reduced IC for quinidine, paroxetine and terbinafine), suggesting morphological changes within the active site cavity that favoured ligand docking and binding. Mutations in CYP2D6*10 and CYP2D6*17 tended to cause deleterious effect on catalysis, with reduced clearance for venlafaxine and CEC. Molecular docking indicated that P34S and T107I, the unique mutations in the alleles, have negatively impacted activity by affecting ligand access and binding due to alteration of the substrate access channel and active site morphology. IC values however were quite variable for quinidine, fluoxetine and terbinafine, and a general decrease in IC was observed for paroxetine, suggesting ligand-specific altered susceptibility to inhibition in the alleles. This study indicates that CYP2D6 allele selectivity for ligands was not solely governed by changes in the active site architecture induced by the mutations, but that the intrinsic properties of the substrates and inhibitors also played vital role.
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http://dx.doi.org/10.1007/s00210-019-01651-0DOI Listing
August 2019

Effect of 95% Ethanol Khat Extract and Cathinone on in vitro Human Recombinant Cytochrome P450 (CYP) 2C9, CYP2D6, and CYP3A4 Activity.

Eur J Drug Metab Pharmacokinet 2019 Jun;44(3):423-431

Department of Biomedical Science, The University of Nottingham Malaysia Campus, Jalan Broga, 43500, Semenyih, Selangor Darul Ehsan, Malaysia.

Background And Objective: A significant number of people worldwide consume khat on daily basis. Long term of khat chewing has shown negative impact on several organ systems. It is likely that these people are co-administered khat preparations and conventional medication, which may lead to khat-drug interactions. This study aimed to reveal the inhibitory potencies of khat ethanol extract (KEE) and its major active ingredient (cathinone) on human cytochrome P450 (CYP) 2C9, CYP2D6, and CYP3A4 enzymes activities, which are collectively responsible for metabolizing 70-80% clinically used drugs.

Methods: In vitro fluorescence-based enzyme assays were developed and the CYP enzyme activities were quantified in the presence and absence of KEE and cathinone employing Vivid CYP450 Screening Kits.

Results: KEE inhibited human CYP2C9, CYP2D6, and CYP3A4 enzyme activities with IC of 42, 62, and 18 μg/ml. On the other hand, cathinone showed negligible inhibitory effect on these CYPs. Further experiments with KEE revealed that KEE inhibited CYP2C9 via non-competitive or mixed mode with K of 14.7 μg/ml, CYP2D6 through competitive or mixed mode with K of 17.6 μg/ml, CYP3A4 by mixed inhibition mode with K of 12.1 μg/ml.

Conclusion: Khat-drug interactions are possible due to administration of clinical drugs metabolized by CYP2C9/CYP2D6/CYP3A4 together with khat chewing. Further in vivo studies are required to confirm our findings and identify the causative constituents of these inhibitory effects.
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http://dx.doi.org/10.1007/s13318-018-0518-2DOI Listing
June 2019

Current High-Throughput Approaches of Screening Modulatory Effects of Xenobiotics on Cytochrome P450 (CYP) Enzymes.

High Throughput 2018 Sep 29;7(4). Epub 2018 Sep 29.

Department of Biomedical Science, the University of Nottingham Malaysia Campus, Jalan Broga, Semenyih 43500, Selangor, Malaysia.

Cytochrome P450 (CYP) is a critical drug-metabolizing enzyme superfamily. Modulation of CYP enzyme activities has the potential to cause drug⁻drug/herb interactions. Drug⁻drug/herb interactions can lead to serious adverse drug reactions (ADRs) or drug failures. Therefore, there is a need to examine the modulatory effects of new drug entities or herbal preparations on a wide range of CYP isoforms. The classic method of quantifying CYP enzyme activities is based on high-performance liquid chromatography (HPLC), which is time- and reagent-consuming. In the past two decades, high-throughput screening methods including fluorescence-based, luminescence-based, and mass-spectrometry-based assays have been developed and widely applied to estimate CYP enzyme activities. In general, these methods are faster and use lower volume of reagents than HPLC. However, each high-throughput method has its own limitations. Investigators may make a selection of these methods based on the available equipment in the laboratory, budget, and enzyme sources supplied. Furthermore, the current high-throughput systems should look into developing a reliable automation mechanism to accomplish ultra-high-throughput screening in the near future.
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http://dx.doi.org/10.3390/ht7040029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6306765PMC
September 2018

The current understanding of the interactions between nanoparticles and cytochrome P450 enzymes - a literature-based review.

Xenobiotica 2019 Jul 12;49(7):863-876. Epub 2018 Sep 12.

c Faculty of Medicine and Health Sciences , Universiti Putra Malaysia , Serdang , Malaysia.

Nanoparticles (NPs) have wide spectrum applications in the areas of industry and biomedicine. However, concerns about their toxic and negative impacts on the environments as well as human health have been raised. Cytochrome P450s (CYPs) are involved in endogenous and exogenous metabolism. Modulations of CYP can adversely damage drug metabolism, detoxification of xenobiotics and animal physiology functions. This article focused on NPs-CYP interactions for humans and animals available in the literature. It was found that different NPs process specific inhibitory potencies against CYPs involved in drug metabolism. Moreover, NPs were able to modify the expression of CYPs genes or protein in humans and other animals, which highlighted their detoxification functions. Nonetheless, changes of CYPs responsible for hormone synthesis and metabolism resulted in endocrine disturbances. Hence, there is a need to screen newly developed NPs to evaluate their interactions with CYPs. The future studies should further strategize the in vitro approaches to reveal the molecular mechanisms behind interactions by taking full considerations of the interference of co-factors, buffers, substrates and metabolites with NPs. Moreover, in vivo studies should compare the influences of NPs via different administration routes and different duration of treatments to reveal the physiological significance.
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http://dx.doi.org/10.1080/00498254.2018.1503360DOI Listing
July 2019

Cytochrome P450 genotype-guided drug therapies: An update on current states.

Clin Exp Pharmacol Physiol 2018 10 2;45(10):991-1001. Epub 2018 Jul 2.

School of Pharmacy, International Medical University, Kuala Lumpur, Malaysia.

Over the past 2 decades, knowledge of the role and clinical value of pharmacogenetic markers has expanded so that individualized pre-emptive therapy based on genetic background of patients could be within reach for clinical implementation. This is evidenced from the frequent updating of drug labels that incorporates pharmacogenetic information (where compelling data become available) by the regulatory agencies (such as the US FDA), and the periodical publication of guidelines of specific therapeutic recommendations based on the results of pharmacogenetic tests by the pharmacogenetics working groups or consortiums of professional bodies. Clinical relevance of the cytochrome P450 (CYP) polymorphism related to dose, effectiveness and/or toxicity of key drugs are presented in this review, including that of warfarin, clopidogrel, tricyclic antidepressants, and proton pump inhibitors. Prospect for routine clinical application of CYP genotyping before prescribing drugs is still currently unclear due to challenges and barriers associated with availability of well-defined and validated pharmacogenetic studies, the interpretation, result reporting and potential error of genotype testing, involvement of non-genetic factors, and other patient's demographic and disease conditions. Further studies to provide additional supporting clinical data and acceleration of pharmacogenetic testing standards and techniques should help improve the evidence base needed for clinical utility and hence move the implementation of genotype-guided therapy in clinical practice a step closer to reality.
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http://dx.doi.org/10.1111/1440-1681.12978DOI Listing
October 2018

Site-Directed Mutagenesis of Cytochrome P450 2D6 and 2C19 Enzymes: Expression and Spectral Characterization of Naturally Occurring Allelic Variants.

Appl Biochem Biotechnol 2018 Sep 10;186(1):132-144. Epub 2018 Mar 10.

School of Pharmacy, International Medical University, 126, Jalan Jalil Perkasa 19, 57000, Bukit Jalil, Kuala Lumpur, Malaysia.

Genetic polymorphism of the cytochrome P450 (CYP) genes particularly affects CYP2D6 and CYP2C19 to a functionally relevant extent, and it is therefore crucial to elucidate the enzyme kinetic and molecular basis for altered catalytic activity of these allelic variants. This study explored the expression and function of the reported alleles CYP2D6*2, CYP2D6*10, CYP2D6*17, CYP2C19*23, CYP2C19*24, and CYP2C19*25 with respect to gene polymorphisms. Site-directed mutagenesis (SDM) was carried out to generate these six alleles. After DNA sequencing, the CYP2D6 and CYP2C19 wild types alongside with their alleles were each independently co-expressed with NADPH-CYP oxidoreductase (OxR) in Escherichia coli. The expressed proteins were analyzed using Western blotting, reduced carbon monoxide (CO) difference spectral scanning, and cytochrome c reductase assay. Results from Western blot revealed the presence of all CYP wild-type and allelic proteins in E. coli membrane fractions. The reduced CO difference spectra scanning presented the distinct peak of absorbance at 450 nm, and the cytochrome c reductase assay has confirmed that spectrally active OxR was expressed in each protein preparation. As a conclusion, the results obtained from this study have proven the CYP variants to be immunoreactive and spectrally active and are suitable for use to examine biotransformation and interaction mechanism of the enzymes.
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http://dx.doi.org/10.1007/s12010-018-2728-0DOI Listing
September 2018

Cytochrome P450 2C9-natural antiarthritic interactions: Evaluation of inhibition magnitude and prediction from in vitro data.

Biopharm Drug Dispos 2018 Apr 23;39(4):205-217. Epub 2018 Mar 23.

School of Pharmacy, International Medical University, Bukit Jalil, Kuala Lumpur, Malaysia.

Many dietary supplements are promoted to patients with osteoarthritis (OA) including the three naturally derived compounds, glucosamine, chondroitin and diacerein. Despite their wide spread use, research on interaction of these antiarthritic compounds with human hepatic cytochrome P450 (CYP) enzymes is limited. This study aimed to examine the modulatory effects of these compounds on CYP2C9, a major CYP isoform, using in vitro biochemical assay and in silico models. Utilizing valsartan hydroxylase assay as probe, all forms of glucosamine and chondroitin exhibited IC values beyond 1000 μM, indicating very weak potential in inhibiting CYP2C9. In silico docking postulated no interaction with CYP2C9 for chondroitin and weak bonding for glucosamine. On the other hand, diacerein exhibited mixed-type inhibition with IC value of 32.23 μM and K value of 30.80 μM, indicating moderately weak inhibition. Diacerein's main metabolite, rhein, demonstrated the same mode of inhibition as diacerein but stronger potency, with IC of 6.08 μM and K of 1.16 μM. The docking of both compounds acquired lower CDOCKER interaction energy values, with interactions dominated by hydrogen and hydrophobic bondings. The ranking with respect to inhibition potency for the investigated compounds was generally the same in both in vitro enzyme assay and in silico modeling with order of potency being diacerein/rhein > various glucosamine/chondroitin forms. In vitro-in vivo extrapolation of inhibition kinetics (using 1 + [I]/K ratio) demonstrated negligible potential of diacerein to cause interaction in vivo, whereas rhein was predicted to cause in vivo interaction, suggesting potential interaction risk with the CYP2C9 drug substrates.
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http://dx.doi.org/10.1002/bdd.2127DOI Listing
April 2018

In vitro and in silico Approaches to Study Cytochrome P450-Mediated Interactions.

J Pharm Pharm Sci 2017 ;20(1):319-328

School of Health Sciences, International Medical University, Jalan Jalil Perkasa, Bukit Jalil, Kuala Lumpur, Malaysia.

In vitro and in silico models of drug metabolism are utilized regularly in the drug research and development as tools for assessing pharmacokinetic variability and drug-drug interaction risk. The use of in vitro and in silico predictive approaches offers advantages including guiding rational design of clinical drug-drug interaction studies, minimization of human risk in the clinical trials, as well as cost and time savings due to lesser attrition during compound development process. This article gives a review of some of the current in vitro and in silico methods used to characterize cytochrome P450(CYP)-mediated drug metabolism for estimating pharmacokinetic variability and the magnitude of drug-drug interactions. Examples demonstrating the predictive applicability of specific in vitro and in silico approaches are described. Commonly encountered confounding factors and sources of bias and error in these approaches are presented. With the advent of technological advancement in high throughput screening and computer power, the in vitro and in silico methods are becoming more efficient and reliable and will continue to contribute to the process of drug discovery, development and ultimately safer and more effective pharmacotherapy. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.
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http://dx.doi.org/10.18433/J3434RDOI Listing
July 2018

Inhibition of Human Cytochrome P450 2c8-catalyzed Amodiaquine N-desethylation: Effect of Five Traditionally and Commonly Used Herbs.

Pharmacognosy Res 2016 Oct-Dec;8(4):292-297

Centre of Excellence for Research in AIDS, Universiti Malaya, 59990 Kuala Lumpur, Malaysia.

Background: In Southeast Asia and many parts of the world, herbal products are increasingly used in parallel with modern medicine.

Objective: This study aimed to investigate the effects of herbs commonly used in Southeast Asia on activity of cytochrome P450 2C8 (CYP2C8), an important human hepatic enzyme in drug metabolism.

Materials And Methods: The selected herbs, such as Jack (ELJ), (LP), (EP), (AP), and (GB), were subjected to inhibition studies using an CYP2C8 activity marker, amodiaquine N-desethylase assay. Inhibition parameters, inhibitory concentration 50% (IC), and K values were determined to study the potency and mode of inhibition.

Results: All herbs inhibited CYP2C8 with the following order of potency: LP > ELJ > GB > AP > EP. LP and ELJ inhibited potently at K's of 2 and 4 times the K of quercetin, the positive control. The inhibition by LP was uncompetitive in nature as compared to competitive or mixed type inhibition observed with other herbs. GB exhibited moderate inhibitory effect at a K6 times larger than quercetin K. AP and EP, on the other hand, showed only weak inhibition.

Conclusion: The herbs we chose represented the more commonly used herbs in Southeast Asia where collision of tradition and modernization in healthcare, if not properly managed, may lead to therapeutic misadventures. We conclude that concurrent consumption of some herbs, in particular, LP and ELJ, may have relevance in drug-herb interactions via CYP2C8 inhibition .

Summary: Herbs are increasingly used in parallel with modern medicines nowadays. In this study five commonly used herbs in Southeast Asia region, ELJ, LP, EP, AP and GB, were investigated for their inhibitory potency on CYP2C8, an important drug-metaboliz-ing human hepatic enzyme. All herbs inhibited CYP2C8 activity marker, amodiaquine N-desethylation, with potency order of LP > ELJ > GB >AP > EP. LP, ELJ and GB exhibited K values of 2, 4 and 6 times the K of quercetin, the positive control, indicating potent to moderate degree of enzyme inhibition. AP and EP, on the other hand, showed only weak inhibition. In summary, concurrent consumption of some herbs especially LP and ELJ may have relevance in drug-herb interactions via CYP2C8 inhibition . : AQ: Amodiaquine, AP: , CYP: Cytochrome P450, DEAQ: Desethylamodiaquine, EP: , ELJ: , GB: , K: Inhibition constant, LP: , Vmax: Maximal velocity, K: Michaelis-Menten constant.
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http://dx.doi.org/10.4103/0974-8490.188886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5004522PMC
October 2016

In Vitro Functional Characterisation of Cytochrome P450 (CYP) 2C19 Allelic Variants CYP2C19*23 and CYP2C19*24.

Biochem Genet 2017 Feb 30;55(1):48-62. Epub 2016 Aug 30.

Department of Biomedical Science, The University of Nottingham Malaysia Campus, Jalan Broga, 43500, Semenyih, Selangor, Malaysia.

Cytochrome P450 (CYP) 2C19 is essential for the metabolism of clinically used drugs including omeprazole, proguanil, and S-mephenytoin. This hepatic enzyme exhibits genetic polymorphism with inter-individual variability in catalytic activity. This study aimed to characterise the functional consequences of CYP2C19*23 (271 G>C, 991 A>G) and CYP2C19*24 (991 A>G, 1004 G>A) in vitro. Mutations in CYP2C19 cDNA were introduced by site-directed mutagenesis, and the CYP2C19 wild type (WT) as well as variants proteins were subsequently expressed using Escherichia coli cells. Catalytic activities of CYP2C19 WT and those of variants were determined by high performance liquid chromatography-based essay employing S-mephenytoin and omeprazole as probe substrates. Results showed that the level of S-mephenytoin 4'-hydroxylation activity of CYP2C19*23 (V 111.5 ± 16.0 pmol/min/mg, K 158.3 ± 88.0 μM) protein relative to CYP2C19 WT (V 101.6 + 12.4 pmol/min/mg, K 123.0 ± 19.2 μM) protein had no significant difference. In contrast, the K of CYP2C19*24 (270.1 ± 57.2 μM) increased significantly as compared to CYP2C19 WT (123.0 ± 19.2 μM) and V of CYP2C19*24 (23.6 ± 2.6 pmol/min/mg) protein was significantly lower than that of the WT protein (101.6 ± 12.4 pmol/min/mg). In vitro intrinsic clearance (CL = V /K for CYP2C19*23 protein was 85.4 % of that of CYP2C19 WT protein. The corresponding CL value for CYP2C19*24 protein reduced to 11.0 % of that of WT protein. These findings suggested that catalytic activity of CYP2C19 was not affected by the corresponding amino acid substitutions in CYP2C19*23 protein; and the reverse was true for CYP2C19*24 protein. When omeprazole was employed as the substrate, K of CYP2C19*23 (1911 ± 244.73 μM) was at least 100 times higher than that of CYP2C19 WT (18.37 ± 1.64 μM) and V of CYP2C19*23 (3.87 ± 0.74 pmol/min/mg) dropped to 13.4 % of the CYP2C19 WT (28.84 ± 0.61 pmol/min/mg) level. Derived from V /K , the CL value of CYP2C19 WT was 785 folds of CYP2C19*23. K and V values could not be determined for CYP2C19*24 due to its low catalytic activity towards omeprazole 5'-hydroxylation. Therefore, both CYP2C19*23 and CYP2C19*24 showed marked reduced activities of metabolising omeprazole to 5-hydroxyomeprazole. Hence, carriers of CYP2C19*23 and CYP2C19*24 allele are potentially poor metabolisers of CYP2C19-mediated substrates.
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http://dx.doi.org/10.1007/s10528-016-9771-8DOI Listing
February 2017

In vitro effect of important herbal active constituents on human cytochrome P450 1A2 (CYP1A2) activity.

Phytomedicine 2014 Oct 16;21(12):1645-50. Epub 2014 Sep 16.

School of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor Darul Ehsan, Malaysia. Electronic address:

This study was designed to investigate eight herbal active constituents (andrographolide, asiaticoside, asiatic acid, madecassic acid, eupatorin, sinensetin, caffeic acid, and rosmarinic acid) on their potential inhibitory effects on human cytochrome P450 1A2 (CYP1A2) activity. A fluorescence-based enzyme assay was performed by co-incubating human cDNA-expressed CYP1A2 with its selective probe substrate, 3-cyano-7-ethoxycoumarin (CEC), in the absence or presence of various concentrations of herbal active constituents. The metabolite (cyano-hydroxycoumarin) formed was subsequently measured in order to obtain IC50 values. The results indicated that only eupatorin and sinensetin moderately inhibited CYP1A2 with IC50 values of 50.8 and 40.2 μM, while the other active compounds did not significantly affect CYP1A2 activity with IC50 values more than 100 μM. Ki values further determined for eupatorin and sinensetin were 46.4 and 35.2 μM, respectively. Our data indicated that most of the investigated herbal constituents have negligible CYP1A2 inhibitory effect. In vivo studies however may be warranted to ascertain the inhibitory effect of eupatorin and sinensetin on CYP1A2 activity in clinical situations.
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http://dx.doi.org/10.1016/j.phymed.2014.08.003DOI Listing
October 2014

Inhibitory potency of 8-methoxypsoralen on cytochrome P450 2A6 (CYP2A6) allelic variants CYP2A6 15, CYP2A6 16, CYP2A6 21 and CYP2A6 22: differential susceptibility due to different sequence locations of the mutations.

PLoS One 2014 27;9(1):e86230. Epub 2014 Jan 27.

Discipline of Pharmacy, Monash University Malaysia, Bandar Sunway, Selangor, Malaysia.

Human cytochrome P450 2A6 (CYP2A6) is a highly polymorphic isoform of CYP2A subfamily. Our previous kinetic study on four CYP2A6 allelic variants (CYP2A6 15, CYP2A6 16, CYP2A6 21 and CYP2A6 22) have unveiled the functional significance of sequence mutations in these variants on coumarin 7-hydroxylation activity. In the present study, we further explored the ability of a typical CYP2A6 inhibitor, 8-methoxypsoralen (8-MOP), in inhibition of these alleles and we hypothesized that translational mutations in these variants are likely to give impact on 8-MOP inhibitory potency. The CYP2A6 variant and the wild type proteins were subjected to 8-MOP inhibition to yield IC50 values. In general, a similar trend of change in the IC50 and Km values was noted among the four mutants towards coumarin oxidation. With the exception of CYP2A6 16, differences in IC50 values were highly significant which implied compromised interaction of the mutants with 8-MOP. Molecular models of CYP2A6 were subsequently constructed and ligand-docking experiments were performed to rationalize experimental data. Our docking study has shown that mutations have induced enlargement of the active site volume in all mutants with the exception of CYP2A6 16. Furthermore, loss of hydrogen bond between 8-MOP and active site residue Asn297 was evidenced in all mutants. Our data indicate that the structural changes elicited by the sequence mutations could affect 8-MOP binding to yield differential enzymatic activities in the mutant CYP2A6 proteins.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0086230PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903516PMC
November 2014

Effect of eurycomanone on cytochrome P450 isoforms CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2E1 and CYP3A4 in vitro.

J Nat Med 2014 Apr 24;68(2):402-6. Epub 2013 Jul 24.

School of Medical Sciences, International Medical University, 126 Jalan 19/155B, Bukit Jalil, 57000, Kuala Lumpur, Malaysia.

Eurycomanone, an active constituent isolated from Eurycoma longifolia Jack, was examined for modulatory effects on cytochrome P450 (CYP) isoforms CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2E1 and CYP3A4 using in vitro assays. The IC50 value was determined to assess the potencies of modulation for each CYP isoform. Our results indicated that eurycomanone did not potently inhibit any of the CYP isoforms investigated, with IC50 values greater than 250 μg/ml. Hence there appears to be little likelihood of drug-herb interaction between eurycomanone or herbal products with high content of this compound and CYP drug substrates via CYP inhibition.
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http://dx.doi.org/10.1007/s11418-013-0794-8DOI Listing
April 2014

In vitro approaches to investigate cytochrome P450 activities: update on current status and their applicability.

Expert Opin Drug Metab Toxicol 2013 Sep 17;9(9):1097-113. Epub 2013 May 17.

Monash University Sunway Campus, Jeffrey Cheah School of Medicine and Health Sciences, Jalan Lagoon Selatan, 46150 Bandar Sunway, Selangor, Malaysia.

Introduction: Cytochromes P450 (CYPs) play a central role in the Phase I metabolism of drugs and other xenobiotics. It is estimated that CYPs can metabolize up to two-thirds of drugs present in humans. Over the past two decades, there have been numerous advances in in vitro methodologies to characterize drug metabolism and interaction involving CYPs.

Areas Covered: This review focuses on the use of in vitro methodologies to examine CYPs' role in drug metabolism and interaction. There is an emphasis on their current development, applicability, advantages and limitations as well as the use of in silico approaches in complementing and supporting in vitro data. The article also highlights the challenges in extrapolating in vitro data to in vivo situations.

Expert Opinion: Advances in in vitro methodologies have been made such that data can be used for in vivo prediction with comfortable degree of confidence. Improved assay designs and analytical techniques have permitted development of miniaturized assay format and automated system with improved sensitivity and throughput capacity. High-quality experimental designs and scientifically rigorous assessment/validation protocols remain crucial in developing reliable and robust in vitro models. With continued progress made in the field, in vitro methodologies will continually be employed in evaluating CYP activities in pharmaceutical industries and laboratories.
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http://dx.doi.org/10.1517/17425255.2013.800482DOI Listing
September 2013

Heterologous expression of human cytochrome P450 (CYP) 2C19 in Escherichia coli and establishment of RP-HPLC method to serve as activity marker.

Biomed Chromatogr 2013 Jul 6;27(7):859-65. Epub 2013 Feb 6.

School of Medical Sciences, No. 126, Jalan Jalil Perkasa 19, Bukit Jalil, 57000, Kuala Lumpur, Malaysia.

In this study, a simple and reliable reverse-phase high-performance liquid chromatography (RP-HPLC) method was established and validated to analyze S-mephenytoin 4-hydroxylase activity of a recombinant CYP2C19 system. This system was obtained by co-expressing CYP2C19 and NADPH-CYP oxidoreductase (OxR) proteins in Escherichia coli (E. coli) cells. In addition to RP-HPLC, the expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. The RP-HPLC assay showed good linearity (r(2) = 1.00) with 4-hydroxymephenytoin concentration from 0.100 to 50.0 μm and the limit of detection was 5.00 × 10(-2) μm. Intraday and interday precisions determined were from 1.90 to 8.19% and from 2.20 to 14.9%, respectively. Recovery and accuracy of the assay were from 83.5 to 85.8% and from 95.0 to 105%. Enzyme kinetic parameters (Km , Vmax and Ki ) were comparable to reported values. The presence of CYP2C19 in bacterial membranes was confirmed by immunoblotting and the characteristic absorbance peak at 450 nm was determined in the reduced CO difference spectral assay. Moreover, the activity level of co-expressed OxR was found to be comparable to that of the literature. As a conclusion, the procedures described here have generated catalytically active CYP2C19 and the RP-HPLC assay developed is able to serve as CYP2C19 activity marker for pharmacokinetic drug interaction study in vitro.
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http://dx.doi.org/10.1002/bmc.2872DOI Listing
July 2013

In-vitro inhibitory effect of Tualang honey on cytochrome P450 2C8 activity.

J Pharm Pharmacol 2012 Dec 8;64(12):1761-9. Epub 2012 Jun 8.

Pharmacogenetics & Novel Therapeutics Cluster, Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia.

Objectives: To investigate the effect of Tualang honey on cytochrome P450 2C8 (CYP2C8) activity in vitro using an amodiaquine N-desethylase assay.

Methods: CYP2C8 and NADPH cytochrome P450 reductase was cotransformed, expressed and harvested. The incubation assay contained expressed proteins, MgCl(2), NADP, glucose 6-phosphate, glucose-6-phosphate dehydrogenase, potassium phosphate buffer, and amodiaquine. The rate of conversion of amodiaquine to desethylamodiaquine, the metabolite, was determined using a high performance liquid chromatography (HPLC) method. The inhibition parameters, IC50 (concentration of inhibitor causing 50% inhibition of original enzyme activity) and apparent inhibition constant (K(i) ) values were determined.

Key Findings: The recombinant proteins were successfully expressed and used to investigate the effect of Tualang honey on CYP2C8 activity. The activity was measured by the rate of metabolism of amodiaquine to desethylamodiaquine determined using a successfully developed HPLC method. Kinetic parameters as determined by nonlinear least-squares regression and evaluated with Aikeike's goodness of fit criteria revealed that Tualang honey competitively inhibited CYP2C8 activity in a dose-dependent manner. Maximum inhibition of 80% occurred at 0.01% honey. The IC50 and K(i) values were (10.0 ± 3.0) × 10(-3) % and (5.1 ± 0.5) × 10(-3) % w/v, respectively.

Conclusions: This study has provided evidence for the in vitro inhibition of CYP2C8-mediated amodiaquine N-desethylase activity by Tualang honey. It revealed that honey, through this inhibition, may have the potential to cause in-vivo drug-food interaction with drugs metabolized by CYP2C8.
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http://dx.doi.org/10.1111/j.2042-7158.2012.01551.xDOI Listing
December 2012

Inhibitory effects of cytochrome P450 enzymes CYP2C8, CYP2C9, CYP2C19 and CYP3A4 by Labisia pumila extracts.

J Ethnopharmacol 2012 Sep 31;143(2):586-91. Epub 2012 Jul 31.

School of Pharmacy and Health Sciences, International Medical University, 126, Jalan 19/155B, Bukit Jalil, 57000 Kuala Lumpur, Malaysia.

Ethnopharmacological Relevance: Labisa pumila (LP), popularly known with its local name, Kacip Fatimah, is a well known herb grown in Indochina and Southeast Asia and is traditionally used to regain energy after giving birth in women. The propensity of LP to cause drug-herb interaction via cytochrome P450 (CYP) enzyme system has not been investigated.

Aim Of The Study: To evaluate the in vitro inhibitory effects of various LP extracts (aqueous, ethanol, dichloromethane (DCM) and hexane) on cytochrome P450 2C8 (CYP2C8), CYP2C9, CYP2C19 and CYP3A4 activities.

Materials And Methods: Probe substrate-based high performance liquid chromatography (HPLC) methods were established for CYP2C9, CYP2C19 and CYP3A4 whereas a fluorescence-based enzyme assay was established for CYP2C8. The metabolite formations were examined after incubation of probe substrate with respective CYP isoform in the present or absent of LP extracts. The inhibitory effect of LP was characterized with kinetic parameters IC(50) and K(i) values.

Results: LP extracts showed differential effect of CYP activities with the order of inhibitory potency as follows: dichloromethane>hexane>ethanol>aqueous. This differential effect was only observed in CYP2C isoforms but not CYP3A4. Both the hexane and DCM extracts exhibited moderate to potent inhibition towards CYP2C activities in different modes including non-competitive, competive and mixed-type. The DCM effect was notably strong for CYP2C8 and CYP2C9 showing K(i) values of below 1 μg/ml. The selectivity of LP for CYP2C isoforms rather than CYP3A4 may be attributed to the presence of relatively small, lipophilic yet slightly polar compounds within the LP extracts.

Conclusions: The results of our study revealed that phytoconstituents contained in LP, particularly in hexane and dichloromethane extracts, were able to selectively inhibit CYP2C isoforms. The inactivation was characterized by low K(i) values, in particular, in CYP2C8 and CYP2C9. These in vitro data indicate that LB preparations contain constituents that can potently inhibit CYP2C activities and suggest that this herb should be examined for potential pharmacokinetic drug interactions in vivo.
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http://dx.doi.org/10.1016/j.jep.2012.07.024DOI Listing
September 2012

In vitro modulatory effects of flavonoids on human cytochrome P450 2C8 (CYP2C8).

Naunyn Schmiedebergs Arch Pharmacol 2012 May 4;385(5):495-502. Epub 2012 Feb 4.

School of Pharmacy and Health Sciences, International Medical University, 126, Jalan Jalil Perkasa 19, Bukit Jalil, 57000, Kuala Lumpur, Malaysia.

The inhibitory effects of five flavonoids with distinct chemical classes (flavones [luteolin], flavonols [quercetin and quercitrin], and flavanones [hesperetin and hespiridin]) on cDNA-expressed CYP2C8 were investigated. CYP2C8 was co-expressed with NADPH-cytochrome P450 reductase in Escherichia coli and used to characterise potency and mechanism of these flavonoids on the isoform. Tolbutamide 4-methylhydroxylase, a high-performance liquid chromatography-based assay, was selected as marker activity for CYP2C8. Our results indicated that the flavonoids inhibited CYP2C8 with different potency. The order of inhibitory activities was quercetin > luteolin > hesperetin > hesperidin > quercitrin. All of these compounds however exhibited mechanism-based inhibition. A number of structural factors were found to be important for inhibition; these include the molecular shape (volume to surface ratio), the number of hydroxyl groups as well as glycosylation of the hydroxyl group. Quercetin was the most potent inhibitor among the flavonoids examined in this study, and our data suggest that it should be examined for potential pharmacokinetic drug interactions pertaining to CYP2C8 substrates in vivo.
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http://dx.doi.org/10.1007/s00210-012-0731-5DOI Listing
May 2012

Heterologous expression of human cytochromes P450 2D6 and CYP3A4 in Escherichia coli and their functional characterization.

Protein J 2011 Dec;30(8):581-91

School of Pharmacy and Health Sciences, International Medical University, 126 Jalan 19/155B, Bukit Jalil, Kuala Lumpur, Malaysia.

This study aimed to express two major drug-metabolizing human hepatic cytochromes P450 (CYPs), CYP2D6 and CYP3A4, together with NADPH-cytochrome P450 oxidoreductase (OxR) in Escherichia coli and to evaluate their catalytic activities. Full length cDNA clones of both isoforms in which the N-terminus was modified to incorporate bovine CYP17α sequence were inserted into a pCWori(+) vector. The modified CYP cDNAs were subsequently expressed individually, each together with OxR by means of separate, compatible plasmids with different antibiotic selection markers. The expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. Enzyme activities were examined using high performance liquid chromatography (HPLC) assays with probe substrates dextromethorphan and testosterone for CYP2D6 and CYP3A4, respectively. Results from immunoblotting demonstrated the presence of both CYP proteins in bacterial membranes and reduced CO difference spectra of the cell preparations exhibited the characteristic absorbance peak at 450 nm. Co-expressed OxR also demonstrated an activity level comparable to literature values. Kinetic parameters, K(m) and V(max) values determined from the HPLC assays also agreed well with literature values. As a conclusion, the procedures described in this study provide a relatively convenient and reliable means of producing catalytically active CYP isoforms suitable for drug metabolism and interaction studies.
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http://dx.doi.org/10.1007/s10930-011-9365-6DOI Listing
December 2011

In vitro determination of the effect of Andrographis paniculata extracts and andrographolide on human hepatic cytochrome P450 activities.

J Nat Med 2011 Jul 3;65(3-4):440-7. Epub 2011 Mar 3.

School of Pharmacy and Health Sciences, International Medical University, 57000, Kuala Lumpur, Malaysia.

We investigated the effects of Andrographis paniculata (AP) extracts and andrographolide on the catalytic activity of three human cDNA-expressed cytochrome P450 enzymes: CYP2C9, CYP2D6 and CYP3A4. In vitro probe-based high performance liquid chromatography assays were developed to determine CYP2C9-dependent tolbutamide methylhydroxylation, CYP2D6-dependent dextromethorphan O-demethylation and CYP3A4-dependent testosterone 6β-hydroxylation activities in the presence and absence of AP extracts and andrographolide. Our results indicate that AP ethanol and methanol extracts inhibited CYP activities more potently than aqueous and hexane extracts across the three isoforms. Potent inhibitory effects were observed on CYP3A4 and CYP2C9 activities (K (i) values below 20 μg/ml). Andrographolide was found to exclusively but weakly inhibit CYP3A4 activity. In conclusion, data presented in this study suggest that AP extracts have the potential to inhibit CYP isoforms in vitro. There was, however, variation in the potency of inhibition depending on the extracts and the isoforms investigated.
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http://dx.doi.org/10.1007/s11418-011-0516-zDOI Listing
July 2011

In vitro effects of active constituents and extracts of Orthosiphon stamineus on the activities of three major human cDNA-expressed cytochrome P450 enzymes.

Chem Biol Interact 2011 Mar 27;190(1):1-8. Epub 2011 Jan 27.

School of Pharmacy and Health Sciences, International Medical University, 126, Jalan 19/155B, Bukit Jalil, 57000 Kuala Lumpur, Malaysia.

Orthosiphon stamineus (OS) has been traditionally used to treat diabetes, kidney and urinary disorders, high blood pressure and bone or muscular pain. To assess the possibility of drug-herb interaction via interference of metabolism, effects of four OS extracts of different polarity and three active constituents (sinensetin, eupatorin and rosmarinic acid) on major human cDNA-expressed cytochrome P450 (CYP) enzymes were investigated. Three substrate-probe based high-performance liquid chromatography (HPLC) assays were established to serve as activity markers for CYP2C9, CYP2D6 and CYP3A4. Our results indicate that OS extracts and constituents exhibited differential modulatory effects on different CYPs. While none of the OS components showed significant inhibition on CYP2C9, eupatorin strongly and uncompetitively inhibited CYP2D6 activity with a K(i) value of 10.2μM. CYP3A4 appeared to be the most susceptible enzyme to OS inhibitory effects. It was moderately inhibited by OS dichloromethane and petroleum ether extract with mixed-type and noncompetitive inhibitions (K(i)=93.7 and 44.9μg/mL), respectively. Correlation study indicated that the inhibition was accounted for by the presence of eupatorin in the extracts. When IC(50) values of these extracts were expressed in volume per dose unit to reflect inhibitory effect at recommended human doses from commercially available products, moderate inhibition was also observed. In addition, CYP3A4 was strongly and noncompetitively inhibited by eupatorin alone, with a K(i) value of 9.3μM. These findings suggest that co-administration of OS products, especially those with high eupatorin content, with conventional drugs may have the potential to cause drug-herb interactions involving inhibition of major CYP enzymes.
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http://dx.doi.org/10.1016/j.cbi.2011.01.022DOI Listing
March 2011

In vitro modulatory effects of Andrographis paniculata, Centella asiatica and Orthosiphon stamineus on cytochrome P450 2C19 (CYP2C19).

J Ethnopharmacol 2011 Jan 18;133(2):881-7. Epub 2010 Nov 18.

School of Pharmacy and Health Sciences, International Medical University, 126 Jalan 19/155B, Bukit Jalil, 57000 Kuala Lumpur, Malaysia.

Ethno Pharmacological Relevance: Andrographis paniculata (AP), Centella asiatica (CA) and Orthosiphon stamineus (OS) are three popular herbs traditionally used worldwide. AP is known for the treatment of infections and diabetes and CA is good for wound healing and healthy skin while OS is usually consumed as tea to treat kidney and urinary disorders. Interaction of these herbs with human cytochrome P450 2C19 (CYP2C19), a major hepatic CYP isoform involved in metabolism of many clinical drugs has not been investigated to date.

Aim Of The Study: In this study, the modulatory effects of various extracts and major active constituents of AP, CA and OS on CYP2C19 activities were evaluated.

Materials And Methods: S-mephenytoin, the CYP2C19 substrate probe, was incubated in the presence or absence of AP, CA and OS components. The changes in the rate of metabolite (hydroxymephenytoin) formation were subsequently determined by a high-performance liquid chromatography (HPLC)-based enzyme assay to characterize the modulatory effects.

Results: Among the herbal extracts studied, AP ethanol extract and CA dichloromethane extract exhibited mixed type inhibition towards CYP2C19 with K(i) values of 67.1 and 16.4 μg/ml respectively; CA ethanol extract and OS petroleum ether extract competitively inhibited CYP2C19 activity (K(i)=39.6 and 41.5 μg/ml respectively). Eupatorin (a major active constituent of OS) was found to significantly inhibit CYP2C19 by mixed type inhibition (K(i)=7.1 μg/ml or 20.6 μM).

Conclusions: It was observed that AP, CA and OS inhibited CYP2C19 activity with varying potency. While weak inhibitory effect was observed with AP, moderate to strong inhibition was observed with CA dichloromethane extract and eupatorin, the major OS constituent. Therefore care should be taken when these CA and OS components are co-administered with CYP2C19 substrates (such as omeprazole, proguanil, barbiturates, citalopram, and diazepam).
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http://dx.doi.org/10.1016/j.jep.2010.11.026DOI Listing
January 2011

In vitro modulatory effects on three major human cytochrome P450 enzymes by multiple active constituents and extracts of Centella asiatica.

J Ethnopharmacol 2010 Jul 8;130(2):275-83. Epub 2010 May 8.

School of Pharmacy and Health Sciences, International Medical University, Bukit Jalil, Kuala Lumpur, Malaysia.

Ethnopharmacological Relevance: Centella asiatica (CA) has been widely cultivated as a vegetable or spice in China, Southeast Asia, India, Sri Lanka, Africa, and Oceanic countries and traditionally used for wound healing and maintaining normal blood pressure.

Aim Of The Study: The present study was carried out to examine the potential modulatory effects of three commercially available active components (asiaticoside, asiatic acid and madecassic acid) and four extracts (aqueous, ethanol, dichloromethane and hexane) of CA on three major cDNA-expressed human cytochrome P450 (CYP) isoforms.

Materials And Methods: High-performance liquid chromatography (HPLC)-based enzyme assays, namely tolbutamide 4-methyhydroxylase, dextromethorphan O-demethylase and testosterone 6beta-hydroxylase assays were developed to probe activities of CYP2C9, CYP2D6 and CYP3A4, respectively. Probe substrates were incubated with or without each active component and extract for each isoform, followed by examination of the kinetics parameters, IC(50) and K(i), to characterize modulatory effects.

Results: CYP2C9 was more susceptible to inhibitory effects by CA extracts compared to CYP2D6 and CYP3A4. Moderate degree of inhibition was observed in ethanol (K(i)=39.1 microg/ml) and dichloromethane (K(i)=26.6 microg/ml) extracts implying potential risk of interaction when CYP2C9 substrates are consumed with CA products. The two extracts however showed negligible inhibition towards CYP2D6 and CYP3A4 (IC(50)'s of 123.3 microg/ml and above). Similarly CA aqueous and hexane extracts did not significantly inhibit all three isoforms investigated (IC(50)'s of 117.9 microg/ml and above). Among the active constituents investigated, asiatic acid and madecassic acid appeared to selectively inhibit CYP2C9 and CYP2D6 more than CYP3A4. Of particular interest is the potent inhibitory effect of asiatic acid on CYP2C9 (K(i)=9.1 microg/ml). This signifies potential risk of interaction when substrates for this isoform are taken together with CA products with high asiatic acid content. Inhibitions of asiatic acid with the other isoforms and that of madecassic acid with all isoforms were only moderate (K(i)'s ranged from 17.2 to 84.4 microg/ml). On the other hand, the IC(50) values for asiaticoside were high (1070.2 microg/ml or above) for all three isoforms, indicating negligible or low potential of this compound to modulate CYP enzymatic activity.

Conclusion: Centella asiatica extracts and active constituents inhibited CYP2C9, CYP2D6 and CYP3A4 activities with varying potency with CYP2C9 being the most susceptible isoform to inhibition. Significant inhibition was observed for asiatic acid and CA ethanol and dichloromethane extracts, implying involvement of semipolar constituents from CA in the effect. This study suggested that CA could cause drug-herb interactions through CYP2C9 inhibition.
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http://dx.doi.org/10.1016/j.jep.2010.05.002DOI Listing
July 2010

Functional characterization of cytochrome P450 2A6 allelic variants CYP2A6*15, CYP2A6*16, CYP2A6*21, and CYP2A6*22.

Drug Metab Dispos 2010 May 5;38(5):745-51. Epub 2010 Feb 5.

School of Pharmacy and Health Sciences, International Medical University, Bukit Jalil, Kuala Lumpur, Malaysia.

Variation in CYP2A6 levels and activity can be attributed to genetic polymorphism and, thus, functional characterization of allelic variants is necessary to define the importance of CYP2A6 polymorphism in humans. The aim of the present study was to investigate the reported alleles CYP2A6*15, CYP2A6*16, CYP2A6*21, and CYP2A6*22, in terms of the functional consequences of their mutations on the enzyme catalytic activity. With use of the wild-type CYP2A6 cDNA as template, site-directed mutagenesis was performed to introduce nucleotide changes encoding K194E substitution in CYP2A6*15, R203S substitution in CYP2A6*16, K476R substitution in CYP2A6*21, and concurrent D158E and L160I substitutions in CYP2A6*22. Upon sequence verification, the CYP2A6 wild-type and mutant constructs were individually coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. A kinetic study using a coumarin 7-hydroxylase assay indicated that CYP2A6*15 exhibited higher V(max) than the wild type, whereas all mutant constructs, except for variant CYP2A6*16, exhibited higher K(m) values. Analysis of the V(max)/K(m) ratio revealed that all mutants demonstrated 0.85- to 1.05-fold differences from the wild type, with the exception of variant CYP2A6*22, which only portrayed 39% of the wild-type intrinsic clearance. These data suggested that individuals carrying the CYP2A6*22 allele are likely to have lower metabolism of CYP2A6 substrate than individuals expressing CYP2A6*15, CYP2A6*16, CYP2A6*21, and the wild type.
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http://dx.doi.org/10.1124/dmd.109.031054DOI Listing
May 2010

Functional role of Ile264 in CYP2C8: mutations affect haem incorporation and catalytic activity.

Drug Metab Pharmacokinet 2008 ;23(3):165-74

School of Pharmacy and Health Sciences, International Medical University, Kuala Lumpur, Malaysia.

The work described in this study aimed to express CYP2C8 wild-type and mutant proteins in bacterial expression system and to use the expressed proteins to investigate the structural and functional consequences of a reported allele CYP2C8(*)4 (carrying Ile264Met substitution) on protein activity. Ile264 was replaced by three different amino acids resulting in three mutant constructs, 2C8I264M, 2C8I264R and 2C8I264D. The presence of isoleucine at position 264 in CYP2C8 was found to be important for proper haem insertion and protein folding; whereas bulkier or charged residues were highly disruptive resulting in inactive proteins with minimum spectral and catalytic activities. This was evidenced from the low levels of Soret peak at 450 nm and negligible levels of tolbutamide methylhydroxylase activity. Kinetic study using paclitaxel indicated that all three mutants exhibited only 9.7 to 35.4% of the activity level observed in the wild-type. In addition, the mutants were more sensitive to proteinase K digestion, indicating a possible alteration of conformation. The combined effects of protein instability and compromised catalytic activity resulted in defective CYP2C8 protein which may have clinical implications in carriers of CYP2C8*4, particularly in terms of their capacity to clear potent drugs and their susceptibility to adverse drug reactions.
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http://dx.doi.org/10.2133/dmpk.23.165DOI Listing
August 2008