Abstract
Mitragynine is a major component isolated from Mitragyna speciosa Korth or kratom, a medicinal plant known for its opiate-like and euphoric properties. Multiple toxicity and fatal cases involving mitragynine or kratom have been reported but the underlying causes remain unclear. P-glycoprotein (P-gp) is a multidrug transporter which modulates the pharmacokinetics of xenobiotics and plays a key role in mediating drug-drug interactions. This study investigated the effects of mitragynine on P-gp transport activity, mRNA, and protein expression in Caco-2 cells using molecular docking, bidirectional assay, RT-qPCR, Western blot analysis, and immunocytochemistry techniques, respectively. Molecular docking simulation revealed that mitragynine interacts with important residues at the nucleotide binding domain (NBD) site of the P-gp structure but not with the residues from the substrate binding site. This was consistent with subsequent experimental work as mitragynine exhibited low permeability across the cell monolayer but inhibited digoxin transport at 10 μM, similar to quinidine. The reduction of P-gp activity in vitro was further contributed by the downregulation of mRNA and protein expression of P-gp. In summary, mitragynine is likely a P-gp inhibitor in vitro but not a substrate. Hence, concurrent administration of mitragynine-containing kratom products with psychoactive drugs which are P-gp substrates may lead to clinically significant toxicity. Further clinical study to prove this point is needed.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Adkins JE, Boyer EW, McCurdy CR (2011) Mitragyna speciosa, a psychoactive tree from Southeast Asia with opioid activity. Curr Top Med Chem 11:1165–1175. https://doi.org/10.2174/156802611795371305
Ahmad K, Aziz Z (2012) Mitragyna speciosa use in the northern states of Malaysia: a cross-sectional study. J Ethnopharmacol 141:446–450. https://doi.org/10.1016/j.jep.2012.03.009
Artursson P, Palm K, Luthman K (2012) Caco-2 monolayers in experimental and theoretical predictions of drug transport. Adv Drug Deliv Rev 64(Supplement):280–289. https://doi.org/10.1016/j.addr.2012.09.005
Aszalos A (2007) Drug-drug interactions affected by the transporter protein, P-glycoprotein (ABCB1, MDR1) II. Clinical aspects. Drug Discov Today 12:838–843. https://doi.org/10.1016/j.drudis.2007.07.021
Badhan R, Penny J (2006) In silico modelling of the interaction of flavonoids with human P-glycoprotein nucleotide-binding domain. Eur J Med Chem 41:285–295. https://doi.org/10.1016/j.ejmech.2005.11.012
Borst P, Elferink RO (2002) Mammalian ABC transporters in health and disease. Annu Rev Biochem 71:537–592. https://doi.org/10.1146/annurev.biochem.71.102301.093055
Boyer EW, Babu KM, Adkins JE, McCurdy CR, Halpern JH (2008) Self-treatment of opioid withdrawal using kratom (Mitragynia speciosa Korth). Addiction 103:1048–1050. https://doi.org/10.1111/j.1360-0443.2008.02209.x
Brewer FK, Follit CA, Vogel PD, Wise JG (2014) In silico screening for inhibitors of P-glycoprotein that target the nucleotide binding domains. Mol Pharmacol 86:716–726. https://doi.org/10.1124/mol.114.095414
Bui K, She F, Zhou D, Butler K, Al-Huniti N, Sostek M (2016) The effect of quinidine, a strong P-glycoprotein inhibitor, on the pharmacokinetics and central nervous system distribution of naloxegol. J Clin Pharmacol 56:497–505. https://doi.org/10.1002/jcph.613
Chittrakarn S, Keawpradub N, Sawangjaroen K, Kansenalak S, Janchawee B (2010) The neuromuscular blockade produced by pure alkaloid, mitragynine and methanol extract of kratom leaves (Mitragyna speciosa Korth.). J Ethnopharmacol 129:344–349. https://doi.org/10.1016/j.jep.2010.03.035
Dolghih E, Bryant C, Renslo AR, Jacobson MP (2011) Predicting binding to P-glycoprotein by flexible receptor docking. PLoS Comput Biol 7:e1002083. https://doi.org/10.1371/journal.pcbi.1002083
Domingo O, Roider G, Stover A, Graw M, Musshoff F, Sachs H, Bicker W (2017) Mitragynine concentrations in two fatalities. Forensic Sci Int 271:e1–e7. https://doi.org/10.1016/j.forsciint.2016.12.020
FDA (2012) Guidance for industry-drug interaction studies-study design, data analysis, implications for dosing, and labeling recommendations. Center for Drug Evaluation and Research, Food and Drug Administration, Rockville
Fenner KS, Troutman MD, Kempshall S, Cook JA, Ware JA, Smith DA, Lee CA (2009) Drug–drug interactions mediated through P-glycoprotein: clinical relevance and in vitro–in vivo correlation using digoxin as a probe drug. Clin Pharmacol Ther 85:173–181. https://doi.org/10.1038/clpt.2008.195
Ferreira LG, Dos Santos RN, Oliva G, Andricopulo AD (2015) Molecular docking and structure-based drug design strategies. Molecules 20:13384–13421. https://doi.org/10.3390/molecules200713384
Ferruzza S, Rossi C, Scarino ML, Sambuy Y (2012) A protocol for differentiation of human intestinal Caco-2 cells in asymmetric serum-containing medium. Toxicol in Vitro 26:1252–1255. https://doi.org/10.1016/j.tiv.2012.01.008
Fromm MF, Kim RB, Stein CM, Wilkinson GR, Roden DM (1999) Inhibition of P-glycoprotein-mediated drug transport: a unifying mechanism to explain the interaction between digoxin and quinidine [see comments]. Circulation 99:552–557
Grandjean-Forestier F, Stenger C, Robert J, Verdier M, Ratinaud M-H (2009) The P-glycoprotein 170: just a multidrug resistance protein or a protean molecule? In: ABC transporters and multidrug resistance. John Wiley & Sons, Inc., pp 15–46. doi:https://doi.org/10.1002/9780470495131.ch1
Grundmann O (2017) Patterns of kratom use and health impact in the US—results from an online survey. Drug Alcohol Depend 176:63–70. https://doi.org/10.1016/j.drugalcdep.2017.03.007
Haslam IS, Jones K, Coleman T, Simmons NL (2008) Induction of P-glycoprotein expression and function in human intestinal epithelial cells (T84). Biochem Pharmacol 76:850–861. https://doi.org/10.1016/j.bcp.2008.07.020
Hassan Z, Muzaimi M, Navaratnam V, Yusoff NHM, Suhaimi FW, Vadivelu R, Vicknasingam BK, Amato D, von Hörsten S, Ismail NIW, Jayabalan N, Hazim AI, Mansor SM, Müller CP (2013) From Kratom to mitragynine and its derivatives: physiological and behavioural effects related to use, abuse, and addiction. Neurosci Biobehav Rev 37:138–151. https://doi.org/10.1016/j.neubiorev.2012.11.012
Hennessy M, Spiers JP (2007) A primer on the mechanics of P-glycoprotein the multidrug transporter. Pharmacol Res 55:1–15. https://doi.org/10.1016/j.phrs.2006.10.007
Henningfield JE, Fant RV, Wang DW (2018) The abuse potential of kratom according the 8 factors of the controlled substances act: implications for regulation and research. Psychopharmacology 235:573–589. https://doi.org/10.1007/s00213-017-4813-4
Hillebrand J, Olszewski D, Sedefov R (2010) Legal highs on the internet. Subst Use Misuse 45:330–340. https://doi.org/10.3109/10826080903443628
Hughes RL (2018) Fatal combination of mitragynine and quetiapine—a case report with discussion of a potential herb-drug interaction. Forensic Sci Med Pathol. https://doi.org/10.1007/s12024-018-0049-9
Jamil MFA, Subki MFM, Lan TM, Majid MIA, Adenan MI (2013) The effect of mitragynine on cAMP formation and mRNA expression of mu-opioid receptors mediated by chronic morphine treatment in SK–N–SH neuroblastoma cell. J Ethnopharmacol 148:135–143. https://doi.org/10.1016/j.jep.2013.03.078
Janchawee B, Keawpradub N, Chittrakarn S, Prasettho S, Wararatananurak P, Sawangjareon K (2007) A high-performance liquid chromatographic method for determination of mitragynine in serum and its application to a pharmacokinetic study in rats. Biomed Chromatogr 21:176–183. https://doi.org/10.1002/bmc.731
Jedlička A, Grafnetterová T, Miller V (2003) HPLC method with UV detection for evaluation of digoxin tablet dissolution in acidic medium after solid-phase extraction. J Pharm Biomed Anal 33:109–115. https://doi.org/10.1016/S0731-7085(03)00226-7
Kapp FG, Maurer HH, Auwarter V, Winkelmann M, Hermanns-Clausen M (2011) Intrahepatic cholestasis following abuse of powdered kratom (Mitragyna speciosa). J Med Toxicol 7:227–231. https://doi.org/10.1007/s13181-011-0155-5
Karinen R, Fosen JT, Rogde S, Vindenes V (2014) An accidental poisoning with mitragynine. Forensic Sci Int 245C:e29–e32. https://doi.org/10.1016/j.forsciint.2014.10.025
Kivistö KT, Niemi M, Fromm MF (2004) Functional interaction of intestinal CYP3A4 and P-glycoprotein. Fundam Clin Pharmacol 18:621–626. https://doi.org/10.1111/j.1472-8206.2004.00291.x
Kong WM, Chik Z, Ramachandra M, Subramaniam U, Aziddin RE, Mohamed Z (2011) Evaluation of the effects of Mitragyna speciosa alkaloid extract on cytochrome P450 enzymes using a high throughput assay. Molecules 16:7344–7356. https://doi.org/10.3390/molecules16097344
König J, Müller F, Fromm MF (2013) Transporters and drug-drug interactions: important determinants of drug disposition and effects. Pharmacol Rev 65:944–966. https://doi.org/10.1124/pr.113.007518
Kronstrand R, Roman M, Thelander G, Eriksson A (2011) Unintentional fatal intoxications with mitragynine and O-desmethyltramadol from the herbal blend Krypton. J Anal Toxicol 35:242–247
Li M, de Graaf IAM, de Jager MH, Groothuis GMM (2015) Rat precision-cut intestinal slices to study P-gp activity and the potency of its inhibitors ex vivo. Toxicol in Vitro 29:1070–1078. https://doi.org/10.1016/j.tiv.2015.04.011
Lim EL, Seah TC, Koe XF, Wahab HA, Adenan MI, Jamil MFA, Majid MIA, Tan ML (2013) In vitro evaluation of cytochrome P450 induction and the inhibition potential of mitragynine, a stimulant alkaloid. Toxicol in Vitro 27:812–824. https://doi.org/10.1016/j.tiv.2012.12.014
Lu J, Wei H, Wu J, Jamil MFA, Tan ML, Adenan MI, Wong P, Shim W (2014) Evaluation of the cardiotoxicity of mitragynine and its analogues using human induced pluripotent stem cell-derived cardiomyocytes. PLoS One 9:e115648. https://doi.org/10.1371/journal.pone.0115648
Manda VK, Avula B, Ali Z, Khan IA, Walker LA, Khan SI (2014) Evaluation of in vitro absorption, distribution, metabolism, and excretion (ADME) properties of mitragynine, 7-hydroxymitragynine, and mitraphylline. Planta Med 80:568–576. https://doi.org/10.1055/s-0034-1368444
Manda VK, Avula B, Dale OR, Ali Z, Khan IA, Walker LA, Khan SI (2017) PXR mediated induction of CYP3A4, CYP1A2, and P-gp by Mitragyna speciosa and its alkaloids. Phytother Res 31:1935–1945. https://doi.org/10.1002/ptr.5942
Matsumoto K, Mizowaki M, Suchitra T, Murakami Y, Takayama H, Sakai SI, Aimi N, Watanabe H (1996a) Central antinociceptive effects of mitragynine in mice: contribution of descending noradrenergic and serotonergic systems. Eur J Pharmacol 317:75–81
Matsumoto K, Mizowaki M, Suchitra T, Takayama H, Sakai S, Aimi N, Watanabe H (1996b) Antinociceptive action of mitragynine in mice: evidence for the involvement of supraspinal opioid receptors. Life Sci 59:1149–1155. https://doi.org/10.1016/0024-3205(96)00432-8
Matsumoto K, Horie S, Ishikawa H, Takayama H, Aimi N, Ponglux D, Watanabe K (2004) Antinociceptive effect of 7-hydroxymitragynine in mice: discovery of an orally active opioid analgesic from the Thai medicinal herb Mitragyna speciosa. Life Sci 74:2143–2155. https://doi.org/10.1016/j.lfs.2003.09.054
Meyer MR, Wagmann L, Schneider-Daum N, Loretz B, de Souza CC, Lehr C-M, Maurer HH (2015) P-glycoprotein interactions of novel psychoactive substances—stimulation of ATP consumption and transport across Caco-2 monolayers. Biochem Pharmacol 94:220–226. https://doi.org/10.1016/j.bcp.2015.01.008
Mikkaichi T, Yoshigae Y, Masumoto H, Imaoka T, Rozehnal V, Fischer T, Okudaira N, Izumi T (2014) Edoxaban transport via P-glycoprotein is a key factor for the drug’s disposition. Drug Metab Dispos 42:520–528. https://doi.org/10.1124/dmd.113.054866
Montanari F, Ecker GF (2015) Prediction of drug-ABC-transporter interaction—recent advances and future challenges. Adv Drug Deliv Rev 86:17–26. https://doi.org/10.1016/j.addr.2015.03.001
Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009) AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 30:2785–2791. https://doi.org/10.1002/jcc.21256
Neerman MF, Frost RE, Deking J (2013) A drug fatality involving kratom. J Forensic Sci 58(Suppl 1):S278–S279. https://doi.org/10.1111/1556-4029.12009
Nelsen JL, Lapoint J, Hodgman MJ, Aldous KM (2010) Seizure and coma following kratom (Mitragynina speciosa Korth) exposure. J Med Toxicol 6:424–426. https://doi.org/10.1007/s13181-010-0079-5
Oga EF, Sekine S, Shitara Y, Horie T (2012) P-glycoprotein mediated efflux in Caco-2 cell monolayers: the influence of herbals on digoxin transport. J Ethnopharmacol 144:612–617. https://doi.org/10.1016/j.jep.2012.10.001
Pan X, Mei H, Qu S, Huang S, Sun J, Yang L, Chen H (2016) Prediction and characterization of P-glycoprotein substrates potentially bound to different sites by emerging chemical pattern and hierarchical cluster analysis. Int J Pharm 502:61–69. https://doi.org/10.1016/j.ijpharm.2016.02.022
Peng Y, Yadava P, Heikkinen AT, Parrott N, Railkar A (2014) Applications of a 7-day Caco-2 cell model in drug discovery and development. Eur J Pharm Sci 56:120–130. https://doi.org/10.1016/j.ejps.2014.02.008
Philipp AA, Wissenbach DK, Weber AA, Zapp J, Maurer HH (2011) Metabolism studies of the kratom alkaloids mitraciliatine and isopaynantheine, diastereomers of the main alkaloids mitragynine and paynantheine, in rat and human urine using liquid chromatography-linear ion trap-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 879:1049–1055. https://doi.org/10.1016/j.jchromb.2011.03.005
Saingam D, Assanangkornchai S, Geater AF, Balthip Q (2013) Pattern and consequences of krathom (Mitragyna speciosa Korth.) use among male villagers in southern Thailand: a qualitative study. Int J Drug Policy 24:351–358. https://doi.org/10.1016/j.drugpo.2012.09.004
Schmidt MM, Sharma A, Schifano F, Feinmann C (2011) “Legal highs” on the net-evaluation of UK-based websites, products and product information. Forensic Sci Int 206:92–97. https://doi.org/10.1016/j.forsciint.2010.06.030
Singh D, Muller CP, Vicknasingam BK (2014) Kratom (Mitragyna speciosa) dependence, withdrawal symptoms and craving in regular users. Drug Alcohol Depend 139:132–137. https://doi.org/10.1016/j.drugalcdep.2014.03.017
Srinivasan B, Kolli AR, Esch MB, Abaci HE, Shuler ML, Hickman JJ (2015) TEER measurement techniques for in vitro barrier model systems. J Lab Autom 20:107–126. https://doi.org/10.1177/2211068214561025
Swogger MT, Hart E, Erowid F, Erowid E, Trabold N, Yee K, Parkhurst KA, Priddy BM, Walsh Z (2015) Experiences of kratom users: a qualitative analysis. J Psychoactive Drugs 47:360–367. https://doi.org/10.1080/02791072.2015.1096434
Szakacs G, Paterson JK, Ludwig JA, Booth-Genthe C, Gottesman MM (2006) Targeting multidrug resistance in cancer. Nat Rev Drug Discov 5:219–234. https://doi.org/10.1038/nrd1984
Tachibana T, Kato M, Takano J, Sugiyama Y (2010) Predicting drug-drug interactions involving the inhibition of intestinal CYP3A4 and P-glycoprotein. Curr Drug Metab 11:762–777. https://doi.org/10.2174/138920010794328922
Tan HK, Muhammad TST, Tan ML (2016) 14-Deoxy-11,12-didehydroandrographolide induces DDIT3-dependent endoplasmic reticulum stress-mediated autophagy in T-47D breast carcinoma cells. Toxicol Appl Pharmacol 300:55–69. https://doi.org/10.1016/j.taap.2016.03.017
Tatum WO, Hasan TF, Coonan EE, Smelick CP (2018) Recurrent seizures from chronic kratom use, an atypical herbal opioid. Epilepsy Behav Case Rep 10:18–20. https://doi.org/10.1016/j.ebcr.2018.04.002
Tay YL, Teah YF, Chong YM, Jamil MFA, Kollert S, Adenan MI, Wahab HA, Döring F, Wischmeyer E, Tan ML (2016) Mitragynine and its potential blocking effects on specific cardiac potassium channels. Toxicol Appl Pharmacol 305:22–39. https://doi.org/10.1016/j.taap.2016.05.022
Thongpradichote S, Matsumoto K, Tohda M, Takayama H, Aimi N, Sakai S, Watanabe H (1998) Identification of opioid receptor subtypes in antinociceptive actions of supraspinally-administered mitragynine in mice. Life Sci 62:1371–1378
Utar Z, Majid MIA, Adenan MI, Jamil MFA, Lan TM (2011) Mitragynine inhibits the COX-2 mRNA expression and prostaglandin E2 production induced by lipopolysaccharide in RAW264.7 macrophage cells. J Ethnopharmacol 136:75–82. https://doi.org/10.1016/j.jep.2011.04.011
Vaalburg W, Hendrikse NH, Elsinga PH, Bart J, van Waarde A (2005) P-glycoprotein activity and biological response. Toxicol Appl Pharmacol 207:257–260. https://doi.org/10.1016/j.taap.2005.03.027
Varma MVS, Kapoor N, Sarkar M, Panchagnula R (2004) Simultaneous determination of digoxin and permeability markers in rat in situ intestinal perfusion samples by RP-HPLC. J Chromatogr B 813:347–352. https://doi.org/10.1016/j.jchromb.2004.09.047
Vicknasingam B, Narayanan S, Beng GT, Mansor SM (2010) The informal use of ketum (Mitragyna speciosa) for opioid withdrawal in the northern states of peninsular Malaysia and implications for drug substitution therapy. Int J Drug Policy 21:283–288. https://doi.org/10.1016/j.drugpo.2009.12.003
Wang Q, Strab R, Kardos P, Ferguson C, Li J, Owen A, Hidalgo IJ (2008) Application and limitation of inhibitors in drug-transporter interactions studies. Int J Pharm 356:12–18. https://doi.org/10.1016/j.ijpharm.2007.12.024
Ward AB, Szewczyk P, Grimard V, Lee CW, Martinez L, Doshi R, Caya A, Villaluz M, Pardon E, Cregger C, Swartz DJ, Falson PG, Urbatsch IL, Govaerts C, Steyaert J, Chang G (2013) Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain. Proc Natl Acad Sci U S A 110:13386–13391. https://doi.org/10.1073/pnas.1309275110
White CM (2018) Pharmacologic and clinical assessment of kratom. Am J Health Syst Pharm 75:261–267. https://doi.org/10.2146/ajhp161035
Wise JG (2012) Catalytic transitions in the human MDR1 P-glycoprotein drug binding sites. Biochemistry 51:5125–5141. https://doi.org/10.1021/bi300299z
Yang C, Zhang T, Li Z, Xu L, Liu F, Ruan J, Liu K, Zhang Z (2013) P-glycoprotein is responsible for the poor intestinal absorption and low toxicity of oral aconitine: in vitro, in situ, in vivo and in silico studies. Toxicol Appl Pharmacol 273:561–568. https://doi.org/10.1016/j.taap.2013.09.030
Zhang W, Ling V (2000) Cell-cycle-dependent turnover of P-glycoprotein in multidrug-resistant cells. J Cell Physiol 184:17–26. https://doi.org/10.1002/(SICI)1097-4652(200007)184:1<17::AID-JCP2>3.0.CO;2-U
Acknowledgements
The authors would like to acknowledge MyBrain15 program and USM fellowship for sponsoring NR.
Author contribution statement
TML, MIA, GK, and SFS conceived and designed research. NR and AA conducted experiments. HAW contributed analytical tools for in silico screening. TML and NR analyzed data. TML wrote the manuscript. All authors read and approved the manuscript.
Funding
This fundamental work was supported in parts by the Fundamental Research Grant Scheme (Ministry of Education Malaysia) and RUI (USM) grant awarded to TML.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
No animals or human were used in this study.
Conflict of interest
The authors declare that they have no conflict of interests.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Supplementary Fig. 1
The structure and identity of the mitragynine confirmed using 1H-NMR and 13C-NMR analysis. (DOCX 20 kb)
Supplementary Fig. 2
(a) The growth inhibition curve of mitragynine and etoposide in Caco-2 cells. Positive values (< 100%) represent growth inhibition and negative values (< 0%) represent cytotoxicity as compared with initial cells plated (T0). Retention time and chromatogram of (b) 20 μM mitragynine) (c) 20 μM of digoxin using optimized HPLC parameters. Linear regression curve of (d) mitragynine (e) digoxin (f) Bidirectional permeability of digoxin in the presence and absence of P-gp inhibitor in Caco-2 cells. All data are presented as mean ± SD of three independent experiments (n = 3). (PNG 1039 kb)
Rights and permissions
About this article
Cite this article
Rusli, N., Amanah, A., Kaur, G. et al. The inhibitory effects of mitragynine on P-glycoprotein in vitro. Naunyn-Schmiedeberg's Arch Pharmacol 392, 481–496 (2019). https://doi.org/10.1007/s00210-018-01605-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00210-018-01605-y