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Artemisia, CYP3A4 and scopoletin.

June 28, 2016 - 16:11 -- Pierre Lutgen

In a pionneering study in 2010, the University of Louvain had studied the anti-inflammatory effect and modulation of cytochrome P450 activities by Artemisia annua tea infusions in human intestinal Caco-2 cells (Melillo de Magalhães P1, et al.,. Food Chem. 2012 Sep 15;134.:864-71). These assays were done on aqueous infusions (3.3/L) of Artemisia annua samples from 7 different origins. For all samples there was a significant reduction in the CYP3A4 activity with the highest reduction down to 37% of the control value for the sample from Luxembourg (registration number MNHNL17732 at the herbarium of Luxembourg). Ketoconazole, a well-known specific and potent CYP3A4 inhibitor used for comparison at 50 µM, completely inhibited the CYP3A4 activity. Artemisinin, rosmarinic and chlorogenic acids tested at plausible intestinal concentrations representative of those found in extracts at 3.3g/L had no effect on the CYP3A4 activity.

More recently similar assays were run at the Vrije Universiteit Brussel (Masterproef. K Lazaridi 2014) in the Laboratory of Pr Kris Demeyer. They used ethanolic extracts at much higher concentrations: 32 gr dried leaves of dried Artemisia plant material extracted by Soxhlet in 1 litre ethanol. They worked with Artemisia plants from different origins, including the species Artemisia abrotanum, Artemisia apiacea, Artemisia pontica, Artemisia herba alba, Artemisia absintium, Artemisia afra. The CYP3A4 inhibition was surprisingly high for all Artemisia samples, up to 6 times higher the for ketoconazol (0.11 µg/mL) or for diluted grapefruit juice. This is surprising because grapefruit juice has the reputation to be the strongest CYP3A4 inhibitor from plant origin. Surprising is also the fact that all Artemisia samples show this strong CYP3A4 inhibition without a particular strength for one of these 7 species.

Cytochrome P450 3A4 (abreviated CYP3A4), is an important enzyme in the body, mainly found in the liver and in the intestine. Its purpose is to oxidize small foreign organic molecules (xenobiotics), such as toxins or drugs, so that they can be removed from the body. It is responsible for the metabolism of more than half of ingested drugs, including artemisinin and its derivatives(Giao & de Vries, 2001; Svensson & Ashton, 1999a). CYP 3A4 enzymes pose a problem in drug development and clinical use of drugs because of interactions with drug disposition and/or drug-drug interactions. A high level of enzyme activity toward a drug causes a decrease in that drugs bioavailability (13). Conversely, drugs, or compounds, that inhibit this enzyme can reduce the normal metabolism of another drug, causing elevated levels and potential toxic side effects.

The inhibitory effect on CYP3A4 is generally measured by the biotransformation and hydroxylation of the drug Midazolam.

The University of Otoga in New Zealand evaluated the inhibition of CYP3A4 by a full range of flavonoids, coumarins and some 20 related compounds. The strongest inhibition is caused by bergapten from grapefruit juice, followed in decreasing order by chrysin, quercetin, naringenin, scopoletin, psoralen, hesperitin, kaempferol (Pin-Chuen Ho et al., J Pharmaceut Sci 2001, 4-3, 217-227). Chrysoplenetin a flavonoid present in Artemisia annua inhibits CYP3A4 and increases the AUC of artemisinin (Shijie Wei et al.,Malar J. 2015; 14: 432. The activity of quercetin (absent in Artemisia annua) is controversial. Some authors even find that it activates CYP3A4 (YJ Chae et al., Planta Med 2016, 82, 121.30). Luteolin strongly activates CYP3A4 (H Dong et al., BMC Biochemistry, 2010, 11 :23). But these effects have all been studied in vitro. The in vivo administration of purified forms of these compounds failed to show an effect. Kaempferol administered to rats did not inhibit CYP3A4 (Ping Li et al., Drug Metabolism and Disposition, 2007, 35, 1203-08). The in vitro inhibitors naringin and quercetin do not contribute to the in vivo inhibition of CYP3A4 either (J Rashid et al., Br J Clin Pharmacol. 1993, 36. 460-3).

The inhibition of CYP 3A4 by essential oils of several plants had already been studied in 2004 in Germany (M Unger, Pharmazeutische Zeitschrift, 13, 2004). At a concentration of 100 µg/ml grapefruit oil inhibited 75%, Eucalyptol 52 % and menthol 5%. Striking in this study is the fact that the inhibition by grapefruit oil is by far stronger than the inhibition by grapefruit juice. The effect of spice constituents on the CYP3A4 was studied in Australia (Wexia Zhang et al., Drug Metabolism and Disposition, 2008, 36, 1283-90). Piperine was already known as a bioavailability enhancer. The following spices were found to inhibit CYP3A4 approximately in this ranking order for a 50% reduction in activity : curcumin (40 µM), 6-gingerol (100 µM), citral (250 µM), d-limonene (400 µM), β-caryophyllene (500 µM), 1,8-cineole (1mM), myrcene (1mM). This study has thus shown that spices have a modulation effect, although modest, on CYP3A4. It may even be prudent to advise against the coadministration of potent drugs together with curcumin.

The inhibitory effect of saturated and polyunsaturated fatty acids on CYP3A4 was studied in Thailand (V Hirunpanich et al., Biol Pharm Bull. 2007, 30, 1586-88). Saturated fatty acids (SFA) like palmitic or stearic acid have no effect. Polyunsaturated fatty acids (PUFA), like linoleic, linolenic, docosahexaeonic acid however have a strong inhibitory effect. This deserves more research as fish oils and omega oils are sold on a large scale as food complements. Plants of the Artemisia family also contain unsaturated fatty acids ranging from 0,3 to 1.7 %. The acids with 18 carbon atoms are more abundant than those with 20 carbons. The predominant acid in all Artemisia species are linoleic and linolenic acid (IS Carvalho et al., Food Science and Technology, 2011,44, 293-298). CYP3A4 enzymes metabolize arachidonic acid and linoleic to several biologically active acids like hydroxyeicosatetraenoic acids (HETEs) (Bylund J et al, J Pharmacol Exp Ther. 1998 , 284, 51-60. The interaction PUFA-CYP3A4 is thus very complex and deserves further study.

In vitro studies performed in human cell cultures and animal models suggest that vitamin E might increase the hepatic production of cytochrome CYP3A4; this could potentially lower the efficacy of any drug metabolized by CYP3A4. (MW Clarke et al., Crit Rev Clin Lab Sci. 2008;45:417-50) Because of the wide popularity and use of vitamin E supplements, further research into potential adverse effects is clearly warranted.

The inhibition of CYP3A4 by grapefruits is mainly related to furanocoumarins (bergamottin, psoralens). Their inhibition potential is equal or stronger than ketoconazole the protypical CYP3A4 inhibitor (K Fukuda et al., Pharmacogenetics 1997, 7, 391-6). When furanocoumarins have been removed from grapefruit juice the in vivo inhibition of CYP3A4 becomes insignificant (MF Paine et al., Am J Clin Nutrit 2006, 83, 1097-105). Furthermore, the inhibition occurs exclusively in the small intestine. The lack of effect of furanocoumarins in hepatic CYP3A4 is exemplified by the lack of effect of intravenously administration. Grapefruit juice consumption leads to excretions of a fluorescent material identified as conjugated scopoletin, The precursor of scopoletin is widely present at different concentrations in commercially available grapefruit juices (Runkel M et al., Eur J Clin Pharmacol. 1996;50(3):225-30). It is thus of interest to investigate which other plants are rich in coumarins, scopoletin for example. There are not many plants where scopoletin has been found and analyzed, with the exception of a few medicinal plants like Morinda citrifolia (Noni), Melia azedirach, Tilia cordata. One of the first plants where scopoletin was detected hundred years ago is Artemisia afra (JA Goodson Biochem J, 1922, 16, 489-93). In fact most plants of the Artemisia family are rich in scopoletin. (Eur J Clin Pharmacol. 1996;50(3):225-30).

Scopoletin can markedly affect the pharmacokinetics of artemisinin and increase the plasma concentration, more than other molecules present in Artemisia like arteannuin-B, artemisinic acid, casticin, chrysoplenol (Chao Zhang et al., Asian Pacific J of Trop Med. June 2016.05.04) Scopoletin in vivo even displays an antimalarial activity which had never been detected in vitro (JI Xiao-Guang et al., Acta Parasitol Med Entomol Sin, 2008, 15198-201). The distribution of scopoletin in murine tissues after oral administration also was studied in China (Yu-Feng Xia et al., J Chin Pharmaceut Sc, 2012, doi : 10.5246). The highest concentrations are found in liver, kidney, stomach and small intestine.

Between patients there is a wide variability in the extent of interaction of inhibitors with CYP3A4. Genetic polymorphisms and allele frequencies contribute to four clinical predominant phenotypes: poor, intermediate, extensive and ultra-rapid metabolizers. See RP Bains in Evolution, Medicine and Public Health 2013, 118-134 for their description. The CYP3A4*3 allele for example has a lower frequency in Africa. Frequencies of xenobiotic-metabolizing polymorphisms had already been found in the Philippines between Filipino, Chinese, Korean, Japanese, Malaysian, Indian and Caucasian populations (R da Guia et al., Current Pharmacogenomics and Personalized Medicine, 2014, 12, 51-55). This may of course have an impact on the treatment of malaria and the asymptomatic load of parasites. It may also have an impact on molecular markers linked to resistance in Plasmodium falciparum as studied in Madrid (A Amor et al., Malaria Journal, 2012, 11:100) on blood samples imported over a period of 8 years from 17 African countries.

Pierre Lutgen

Comments

Submitted by Marc Vanacker (not verified) on

If the hypothesis of Dr Lutgen is correct i.e. that Artemisia plants inhibit CYP3A4 and increase the therapeutic efficacy of xenobiotics and other molecules against malaria, than the widespread use of pyrethroids should be looked at with new scrutiny. A surge in the morbidity and mortality of malaria has been noticed over recent years in several countries: Uganda, Burundi, Cameroon, RDCongo, Senegal, India, Ethiopia, Peru…In Guinea-Bissau the increase was related to the massive introduction of pyrethroid impregnated ITNs in 2010 (J Ursing et al., PLoS ONE 9 e101167). It is also suspected that the indirect effects of ITNs on clinical immunity might be more substantial with the deployment of ACTs than with chloroquine, causing a rebound and age shift of malaria at Dielmo in Senegal (JF Trape et al, The Lancet, 11 Dec 2011).

In 2008 already a study had shown that deltamethrin caused a 25-fold increase in CYP3A4 in human hepatocytes and permethrin a 4-fold increase (PC Das et al., Drug Metab Drug Interact 2008, 23, 211-36). Similar interactions in the metabolism of pyrethroids were measured in another study and the authors recommended that the potential adverse health effects of these pesticides should not be neglected. Due to their lipophilicity, pyrethroids are absorbed through the gastrointestinal and respiratory tracts and are preferentially stored in lipid-rich body fat and nerve tissues (EJ Scollon et al., Drug Metab Dispos 2009, 37, 221-8). A more recent study evaluated the impact of 24 pesticides on CYP3A4 (K Abass et al., Toxicology, 2012 Mar 29, 17-26). CYP3A4 was induced most powerfully by pyrethroids: a 35-fold increase for 50 µM cypermethrin.

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