Artemisinin Metabolism Research Articles

AaMYC3 bridges the regulation of glandular trichome density and artemisinin biosynthesis in Artemisia annua.

Artemisinin, the well-known natural product for treating malaria, is biosynthesised and stored in the glandular-secreting trichomes (GSTs) of Artemisia annua. While numerous efforts have clarified artemisinin metabolism and regulation, the molecular association between artemisinin biosynthesis and GST development remains elusive. Here, we identified AaMYC3, a bHLH transcription factor of A. annua, induced by jasmonic acid (JA), which simultaneously regulates GST density and artemisinin biosynthesis. Overexpressing AaMYC3 led to a substantial increase in GST density and artemisinin accumulation. Conversely, in the RNAi-AaMYC3 lines, both GST density and artemisinin content were markedly reduced. Through RNA-seq and analyses conducted both in vivo and in vitro, AaMYC3 not only directly activates AaHD1 transcription, initiating GST development, but also up-regulates the expression of artemisinin biosynthetic genes, including CYP71AV1 and ALDH1, thereby promoting artemisinin production. Furthermore, AaMYC3 acts as a co-activator, interacting with AabHLH1 and AabHLH113, to trigger the transcription of two crucial enzymes in the artemisinin biosynthesis pathway, ADS and DBR2, ultimately boosting yield. Our findings highlight a critical connection between GST initiation and artemisinin biosynthesis in A. annua, providing a new target for molecular design breeding of traditional Chinese medicine.

Drug-Drug Interactions of Artemisinin-Based Combination Therapies in Malaria Treatment: A Narrative Review of the Literature.

Artemisinin is an antimalarial compound derived from the plant Artemisia annua L., also known as sweet wormwood. According to the World Health Organization, artemisinin-based combination therapy (ACT) is an essential treatment for malaria, specifically Plasmodium falciparum, which accounts for most malaria-related mortality. ACTs used to treat uncomplicated malaria include artemether-lumefantrine, artesunate-amodiaquine, artesunate-mefloquine, artesunate-sulphadoxine-pyrimethamine, and dihydroartemisinin-piperaquine. Although the mechanism of action and clinical capabilities of artemisinin in malaria treatment are widely known, more information on the potential for drug interactions needs to be further investigated. Some studies show pharmacokinetic and pharmacodynamic drug interactions with HIV antiviral treatment but few studies have been conducted on most other drug classes. Based on known genotypes of cytochrome P450 (CYP) enzymes, CYP2B6 and CYP3A are primarily involved in the metabolism of artemisinin and its derivatives. Reduced functions in these enzymes can lead to subtherapeutic concentrations of the active metabolite, dihydroartemisinin, that may cause treatment failure, which has been shown in some studies with cardiovascular, antibiotic, and antiparasitic drugs. Although the clinical importance remains unclear to date, clinicians should be aware of potential drug-drug interactions and monitor patients on ACT closely.

In Vitro Adventitious Regeneration of Artemisia annua L. Influencing Artemisinin Metabolism

Artemisia annua L. is a herbaceous plant belonging to the Asteraceae family, known for producing, although at low levels, the sesquiterpene lactone artemisinin (AN), which is highly effective against malaria. In this study, an in vitro regeneration process of A. annua L. using ‘Artemis’ progeny was established and the potential of tissue culture for inducing new variability in terms of AN metabolism of in vitro regenerated plants was investigated. Among the plant growth regulators tested, the cytokinin 6-benzyladenine (BA) at 4.4 μM in combination with the auxin indole-butyric acid (IBA) at 0.35 μM yielded the greatest frequency of shoot induction. The optimal multiplication medium contained BA at 0.9 μM and naphthaleneacetic acid (NAA) at 0.05 μM. Regenerated plants (RPs), after transferring to the greenhouse and subsequently to the field, were analyzed during the growth cycle at different sampling times, showing a peak of AN content 20 days before blossom. Variability among different RPs and sampling times, in terms of AN and its precursors dihydroartemisinic acid (DHAA) and artemisinic acid (AA) was observed. This suggests that adventitious shoot induction could provide a useful strategy to induce variability influencing artemisinin metabolism as a consequence of in vitro manipulation.

Characterization of a class III peroxidase from Artemisia annua: relevance to artemisinin metabolism and beyond.

A class III peroxidase from Artemisia annua has been shown to indicate the possibility of cellular localization-based role diversity, which may have implications in artemisinin catabolism as well as lignification. Artemisia annua derives its importance from the antimalarial artemisinin. The -O-O- linkage in artemisinin makes peroxidases relevant to its metabolism. Earlier, we identified three peroxidase-coding genes from A. annua, whereby Aa547 showed higher expression in the low-artemisinin plant stage whereas Aa528 and Aa540 showed higher expression in the artemisinin-rich plant stage. Here we carried out tertiary structure homology modelling of the peroxidases for docking studies. Maximum binding affinity for artemisinin was shown by Aa547. Further, Aa547 showed greater binding affinity for post-artemisinin metabolite, deoxyartemisinin, as compared to pre-artemisinin metabolites (dihydroartemisinic hydroperoxide, artemisinic acid, dihydroartemisinic acid). It also showed significant binding affinity for the monolignol, coniferyl alcohol. Moreover, Aa547 expression was related inversely to artemisinin content and directly to total lignin content as indicated by its transient silencing and overexpression in A. annua. Artemisinin reduction assay also indicated inverse relationship between Aa547 expression and artemisinin content. Subcellular localization using GFP fusion suggested that Aa547 is peroxisomal. Nevertheless, dual localization (intracellular/extracellular) of Aa547 could not be ruled out due to its effect on both, artemisinin and lignin. Taken together, this indicates possibility of localization-based role diversity for Aa547, which may have implications in artemisinin catabolism as well as lignification in A. annua.

The Prevalence of CYP2B6 Gene Polymorphisms in Malaria-endemic Population of Timor in East Nusa Tenggara Indonesia.

ObjectivesThe CYP2B6 is one of the most polymorphic CYP genes in humans that has the potential to modify the pharmacological and toxicological responses to clinically important drugs such as antimalarial artemisinin and its derivatives. The aim of the study was to determine the frequency of CYP2B6 polymorphisms in Timor malaria endemic area, East Nusa Tenggara, Indonesia where Artemisin-based Combination Therapy (ACT) has been used to treat uncomplicated malaria.MethodsA total of 109 healthy subjects were participated in this study. CYP2B6*4, *6 and *9 polymorphisms were analyzed using PCR-RFLP to confirm the SNPs prevalence of 516G>T and 785A>G in exon 4 and 5.ResultsThere were 96 subjects included in the analysis. In the exon 4 of CYP2B6 516G>T, the frequency of the T mutation was 37.5% (39/96), and the wildtype 27.1% (26/96). In the exon 5, CYP2B6 785A>G mutant was detected in 29.2% (28/96) of individuals, and the wildtype allele in 35.4% (34/96). The frequency of CYP2B6*9 (516G>T), CYP2B6*4 (785A>G) and CYP2B6*6 (516G>T and 785A>G) were 40.6%, 29.2% and 22.9%, respectively. The prevalence of these CYP2B6 gene polymorphisms in Timorian ethnic were higher than that in Malay, Han Chinese, Indian, and Egyptian populations.ConclusionThe prevalence of these CYP2B6 516G>T and 785A>G polymorphisms in Timorian ethnic is higher than that in other populations. These polymorphisms may affect the metabolism of artemisinin and its derivatives.

Differentially Expressed Peroxidases from Artemisia annua and Their Responses to Various Abiotic Stresses

Artemisia annua is well-known for producing the antimalarial phytomolecule, artemisinin. The role of peroxidases has been hypothesized in artemisinin metabolism owing to the presence of an –O–O– linkage in this sesquiterpene lactone. Earlier, using a microarray, we identified differentially expressed genes, including peroxidases, in plant growth stages having contrasting artemisinin content. Here, three peroxidases—Aa547, having higher expression in low-artemisinin stage, and Aa540 and Aa528, having higher expression in high artemisinin stage, which could be associated with trichomes on the basis of their approximate gene expression pattern inferred from EST counts in UniGene—were selected for full-length cloning, tissue-specific expression profiling, and in silico analyses. The upstream genomic region of Aa547 was cloned and various cis-regulatory elements were identified. All the three candidates were predicted to be class III plant peroxidases. Further, this study aimed to check the responsiveness of the logically selected peroxidase genes to various abiotic stress factors. Taking cues from previous reports and the regulatory elements observed in the Aa547 promoter, hydration, salinity, temperature, salicylic acid, hydrogen peroxide, and methyl jasmonate, were selected to study their effect on the expression of the peroxidase genes through qRT-PCR. The peroxidases were found to be highly sensitive to the various factors but differed in their responses. Broadly, except for responses to high temperature and salicylic acid, the response of Aa547 to various factors was distinct from that of Aa540 and Aa528, which was in line with its distinctness from the other two peroxidases, considering the in planta artemisinin content and predicted structural features.

Auto-induction of phase I and phase II metabolism of artemisinin in healthy Chinese subjects after oral administration of a new artemisinin-piperaquine fixed combination.

BackgroundArtequick is a relatively inexpensive artemisinin (Qing-hao-su; QHS)-based combination therapy (ACT) that contains QHS and piperaquine (PQ), which has not been widely used because of the decreased concentration level of QHS after repeated oral administrations for five to seven days as a monotherapy. This study was designed to evaluate the potential auto-induction metabolism of QHS in healthy Chinese adults after a two-day oral administration of QHS-PQ. The effect of QHS-PQ on the activity of the CYP2B6 and CYP3A4 was also investigated.MethodsFourteen healthy Chinese subjects received two-day oral doses of QHS-PQ (Artequick). A two-drug cocktail consisting of bupropion and midazolam was used to assess the activities of CYP2B6 and CYP3A, respectively. Plasma samples were analysed for QHS and its phase I/II metabolites, probe drugs and their metabolites, using a validated liquid chromatography tandem mass spectrometric (LC-MS) method.ResultsFour major phase I metabolites of QHS (M1-M3 and deoxy-QHS) and two subsequent phase II metabolites (M4-M5) were detected in human plasma after oral administrations of QHS-PQ. The AUC 0-t of the QHS and its phase I metabolites decreased significantly (P < 0.05) with increased oral clearance (CL/F) after two-day oral doses of QHS-PQ, whereas its phase II metabolites exhibited higher AUC (P < 0.01). The phase I metabolic capability, calculated by the AUC 0-t ratio of all phase I metabolites to QHS, increased 1.5-fold after the repeated dose (P < 0.01), and the phase II metabolic capability increased 1.5-fold for M4 and 3.0-fold for M5. The enzyme activity of CYP2B6 and CYP3A4 increased 2.1-fold and 3.2-fold, respectively, after two-day oral doses of QHS-PQ.ConclusionsThe auto-induction of both phase I and phase II metabolism of QHS was present in healthy Chinese subjects after a recommended two-day oral dose of QHS-PQ. The auto-induction metabolism also existed for phase I metabolites of QHS. The enzyme activity of CYP2B6 and CYP3A4 was induced after the two-day oral doses of QHS-PQ. Based on these results, the alternative common three-day regimen for QHS-PQ could probably lead to lower bioavailability of QHS and higher potential of drug-drug interaction caused by the induction of drug-metabolizing enzymes.

Pharmacokinetics of artemisinin delivered by oral consumption of Artemisia annua dried leaves in healthy vs. Plasmodium chabaudi-infected mice

Ethnopharmacological relevanceThe Chinese have used Artemisia annua as a tea infusion to treat fever for >2000 years. The active component is artemisinin. Previously we showed that when compared to mice fed an equal amount of pure artemisinin, a single oral dose of dried leaves of Artemisia annua (pACT) delivered to Plasmodium chabaudi-infected mice reduced parasitemia at least fivefold. Dried leaves also delivered >40 times more artemisinin in the blood with no toxicity. The pharmacokinetics (PK) of artemisinin delivered from dried plant material has not been adequately studied. Materials and methodsHealthy and Plasmodium chabaudi-infected mice were oral gavaged with pACT to deliver a 100mgkg−1 body weight dose of artemisinin. Concentrations of serum artemisinin and one of its liver metabolites, deoxyartemisinin, were measured over two hours by GCMS. ResultsThe first order elimination rate constant for artemisinin in pACT-treated healthy mice was estimated to be 0.80h−1 with an elimination half-life (T½) of 51.6min. The first order absorption rate constant was estimated at 1.39h−1. Cmax and Tmax were 4.33mgL−1 and 60min, respectively. The area under the curve (AUC) was 299.5mgminL−1. In contrast, the AUC for pACT-treated infected mice was significantly greater at 435.6mgminL−1. Metabolism of artemisinin to deoxyartemisinin was suppressed in infected mice over the period of observation. Serum levels of artemisinin in the infected mice continued to rise over the 120min of the study period, and as a result, the T½ was not determined; the Cmax and Tmax were estimated at ≥6.64mgL−1 and ≥120min, respectively. Groups of healthy mice were also fed either artemisinin or artemisinin mixed in mouse chow. When compared at 60min, artemisinin was undetectable in the serum of mice fed 100mgANkg−1 body weight. When plant material was present either as mouse chow or Artemisia annua pACT, artemisinin levels in the serum rose to 2.44 and 4.32mgL−1, respectively, indicating that the presence of the plant matrix, even that of mouse chow, had a positive impact on the appearance of artemisinin in the blood. ConclusionsThese results showed that artemisinin and one of its drug metabolites were processed differently in healthy and infected mice. The results have implications for possible therapeutic use of pACT in treating malaria and other artemisinin-susceptible diseases.

Differentially Expressed Genes during Contrasting Growth Stages of Artemisia annua for Artemisinin Content

Artemisia annua is the source of antimalarial phytomolecule, artemisinin. It is mainly produced and stored in the glandular secretory trichomes present in the leaves of the plant. Since, the artemisinin biosynthesis steps are yet to be worked out, in this investigation a microarray chip was strategized for the first time to shortlist the differentially expressing genes at a stage of plant producing highest artemisinin compared to the stage with no artemisinin. As the target of this study was to analyze differential gene expression associated with contrasting artemisinin content in planta and a genotype having zero/negligible artemisinin content was unavailable, it was decided to compare different stages of the same genotype with contrasting artemisinin content (seedling - negligible artemisinin, mature leaf - high artemisinin). The SCAR-marked artemisinin-rich (∼1.2%) Indian variety ‘CIM-Arogya’ was used in the present study to determine optimal plant stage and leaf ontogenic level for artemisinin content. A representative EST dataset from leaf trichome at the stage of maximal artemisinin biosynthesis was established. The high utility small scale custom microarray chip of A. annua containing all the significant artemisinin biosynthesis-related genes, the established EST dataset, gene sequences isolated in-house and strategically selected candidates from the A. annua Unigene database (NCBI) was employed to compare the gene expression profiles of two stages. The expression data was validated through semiquantitative and quantitative RT-PCR followed by putative annotations through bioinformatics-based approaches. Many candidates having probable role in artemisinin metabolism were identified and described with scope for further functional characterization.

Autoinduction of phase I and phase II metabolism of artemisinin in rats

This study was designed to get the direct evidence of the autoinduction metabolism for the antimalarial drug artemisinin (QHS). The sex effect on the pharmacokinetic profiles of QHS and its metabolites was also studied.Two groups of rats received a single oral dose of QHS, and another two groups of rats were given oral doses of QHS once daily for 5 consecutive days. Plasma samples and its phase I and phase II metabolites were analysed for QHS, using a validated liquid chromatography tandem mass spectrometric (LC-MS) method.Eight phase I metabolites (DQHS, M1–M7) and five phase II metabolites (M8–M12) of QHS were detected in rat plasma. The AUC0−t of the parent drug QHS, and its phase I metabolites DQHS, M2, M3 and M6 decreased significantly (p < 0.05) with increased oral clearance (CL/F) (p < 0.05) after 5-day oral doses of QHS to rats. There was no change (p > 0.05) in AUC of M1 and M4, whereas its metabolites M5 and M7 exhibited higher AUC (p < 0.05). The AUC of phase II metabolites M8, M11 and M12 also increased after multiple oral doses of QHS. Sex difference was observed for QHS and its metabolites DQHS, M1, M3, M5, M8 and M9 in rats after a single oral dose of QHS.The results gave the direct evidence for the autoinduction of both phase I and phase II metabolism of QHS. The sex effect existed for QHS.

Flavonoids from Artemisia annua L. as Antioxidants and Their Potential Synergism with Artemisinin against Malaria and Cancer

Artemisia annua is currently the only commercial source of the sesquiterpene lactone artemisinin. Since artemisinin was discovered as the active component of A. annua in early 1970s, hundreds of papers have focused on the anti-parasitic effects of artemisinin and its semi-synthetic analogs dihydroartemisinin, artemether, arteether, and artesunate. Artemisinin per se has not been used in mainstream clinical practice due to its poor bioavailability when compared to its analogs. In the past decade, the work with artemisinin-based compounds has expanded to their anti-cancer properties. Although artemisinin is a major bioactive component present in the traditional Chinese herbal preparations (tea), leaf flavonoids, also present in the tea, have shown a variety of biological activities and may synergize the effects of artemisinin against malaria and cancer. However, only a few studies have focused on the potential synergistic effects between flavonoids and artemisinin. The resurgent idea that multi-component drug therapy might be better than monotherapy is illustrated by the recent resolution of the World Health Organization to support artemisinin-based combination therapies (ACT), instead of the previously used monotherapy with artemisinins. In this critical review we will discuss the possibility that artemisinin and its semi-synthetic analogs might become more effective to treat parasitic diseases (such as malaria) and cancer if simultaneously delivered with flavonoids. The flavonoids present in A. annua leaves have been linked to suppression of CYP450 enzymes responsible for altering the absorption and metabolism of artemisinin in the body, but also have been linked to a beneficial immunomodulatory activity in subjects afflicted with parasitic and chronic diseases.

Global characterization of Artemisia annua glandular trichome transcriptome using 454 pyrosequencing

BackgroundGlandular trichomes produce a wide variety of commercially important secondary metabolites in many plant species. The most prominent anti-malarial drug artemisinin, a sesquiterpene lactone, is produced in glandular trichomes of Artemisia annua. However, only limited genomic information is currently available in this non-model plant species.ResultsWe present a global characterization of A. annua glandular trichome transcriptome using 454 pyrosequencing. Sequencing runs using two normalized cDNA collections from glandular trichomes yielded 406,044 expressed sequence tags (average length = 210 nucleotides), which assembled into 42,678 contigs and 147,699 singletons. Performing a second sequencing run only increased the number of genes identified by ~30%, indicating that massively parallel pyrosequencing provides deep coverage of the A. annua trichome transcriptome. By BLAST search against the NCBI non-redundant protein database, putative functions were assigned to over 28,573 unigenes, including previously undescribed enzymes likely involved in sesquiterpene biosynthesis. Comparison with ESTs derived from trichome collections of other plant species revealed expressed genes in common functional categories across different plant species. RT-PCR analysis confirmed the expression of selected unigenes and novel transcripts in A. annua glandular trichomes.ConclusionThe presence of contigs corresponding to enzymes for terpenoids and flavonoids biosynthesis suggests important metabolic activity in A. annua glandular trichomes. Our comprehensive survey of genes expressed in glandular trichome will facilitate new gene discovery and shed light on the regulatory mechanism of artemisinin metabolism and trichome function in A. annua.

Artemisinin–a possible CYP2B6 probe substrate?

To compare in vitro metabolism rates for artemisinin and the CYP2B6 substrates, bupropion, propofol and efavirenz in human liver microsomes. Rate constants of artemisinin, bupropion, propofol and efavirenz metabolism by human liver microsomes from a panel of 12 donors, with different levels of CYP2B6 activity, were estimated in WinNonlin. Correlations between the metabolic rate constant for artemisinin and the other CYP2B6 substrates were examined. Artemisinin and propofol depletion data in human liver microsomes were described by first order kinetic models. For bupropion and efavirenz, metabolite formation data were incorporated in the model. Rate constants varied considerably for all substrates. There was a high degree of correlation of rate constants between substrates (r> or =0.87, p<0.001). The rate of in vitro metabolism of artemisinin was correlated significantly to that of bupropion, propofol and efavirenz, suggesting artemisinin to be a potential alternative marker to assess CYP2B6 activity. Further studies characterizing the metabolic fate of artemisinin are needed in order to evaluate its utility as an in vitro and in vivo CYP2B6 probe substrate, since CYP2B6 might not be the only CYP isoform involved in the depletion of artemisinin.

Secondary Metabolic Profiling and Artemisinin Biosynthesis of Two Genotypes ofArtemisia annua

Artemisinin has been proven to be an effective antimalarial compound, especially for chloroquine-resistant and cerebral malaria. However, its biosynthesis pathway is still not completely clear. In order to get new clues about artemisinin biosynthesis, metabolic profiling by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) was applied to compare the secondary metabolites of two Artemisia annua L., genotype SP18 and 001, for some phenotypic and agricultural trait differences, including artemisinin content, existed between the two genotypes. Samples at 7 time points of three growth stages were studied. The data of profiles were subjected to multivariate analysis with partial least squares discriminant analysis (PLS-DA). The results indicated that there were clear differences in terpenoids and artemisinin metabolism between different growth stages and genotypes. Twenty-one compounds, including artemisinin and its related precursors, were selected as the marker compounds of the PLS-DA between the two genotypes. Among them, artemisinic acid, arteannuin B, borneol, beta-farnesene and an unidentified sesquiterpenoid (peak 48) were abundant in 001, while camphor, methyl artemisinic acid and lanceol accumulated mainly in SP18. The relationship between these differences and artemisinin biosynthesis in the two genotypes of A. annua were discussed.

Retrospective analysis of artemisinin pharmacokinetics: application of a semiphysiological autoinduction model

To describe the time-course of the autoinduction of artemisinin by applying a semi-physiological pharmacokinetic model. Plasma concentration-time data from six clinical studies involving oral administration of artemisinin to healthy subjects and malaria patients were included in the analysis. NONMEM was used to apply a semi-physiological model incorporating metabolizing enzymes and a pharmacokinetic model including a separate hepatic compartment. The model described the data well. The hepatic extraction ratio increased from 0.74 at pre-induced conditions to 0.98 after autoinduction of metabolism. Our model successfully described the time-course of autoinduction of metabolism of artemisinin in subjects receiving oral artemisinin.

In vitro evidence for auto-induction of artemisinin metabolism in the rat

Artemisinin disappearance rate was more rapid in incubations with liver microsomes from rats pre-treated with oral artemisinin (60 mg/kg/day for 5 days) compared with microsomes from control animals. A single pathway Michaelis-Menten saturable elimination model was fitted to the concentration-time data of artemisinin incubations by non-linear regression. Model parameters were obtained after fitting results for each animal separately and by pooling data for pre-treated and control animals. Parameter estimates (% coefficient of variation) from fitting the pooled data was maximum velocities (Vmax) = 1.8 (12) mmole/min/mg protein and Michaelis constants (Km) = 20(22) microM for artemisinin pre-treated and Vmax = 0.85 (35) mmole/min/mg protein and Km = 67(52) microM for control animals indicating a 2-fold increase in Vmax and a 3-fold decrease in Km with microsomes from artemisinin pre-treated animals. Estimates of intrinsic clearance in microsomes from the pre-treated animals were 8-fold higher compared with controls. Thus, artemisinin appears to be a potent auto-inducer of drug metabolism in rats as has also been observed in humans. The present findings suggest caution in the interpretation of repeat-dose rat toxicity studies with artemisinin unless its pharmacokinetics are simultaneously monitored, since during multiple administration, the exposure of the drug will not be constant over time.

Pharmacokinetic interactions of antimalarial agents.

Combination of antimalarial agents has been introduced as a response to widespread drug resistance. The higher number of mutations required to express complete resistance against combinations may retard the further development of resistance. Combination of drugs, especially with the artemisinin drugs, may also offer complete and rapid eradication of the parasite load in symptomatic patients and thus reduce the chance of survival of resistant strains. The advantages of combination therapy should be balanced against the increased chance of drug interactions. During the last decade, much of the pharmacokinetics and metabolic pathways of antimalarial drugs have been elucidated, including the role of the cytochrome P450 (CYP) enzyme complex. Change in protein binding is not a significant cause of interactions between antimalarial agents. CYP3A4 and CYP2C19 are frequently involved in the metabolism of antimalarial agents. Quinidine is a potent inhibitor of CYP2D6, but it appears that this enzyme does not mediate the metabolism of any other antimalarial agent. The new combinations proguanil-atovaquone and chlorproguanil-dapsone do not show significant interactions. CYP2B6 and CYP3A4 are involved in the metabolism of artemisinin and derivatives, but further studies may reveal involvement of more enzymes. Artemisinin may induce CYP2C19. Several artemisinin drugs suffer from auto-induction of the first-pass effect, resulting in a decline of bioavailability after repeated doses. The mechanism of this effect is not yet clear, but induction by other agents cannot be excluded. The combination of artemisinin drugs with mefloquine and the fixed combination artemether-lumefantrine have been studied widely, and no significant drug interactions have been found. The artemisinin drugs will be used at an increasing rate, particularly in combination with other agents. Although clinical studies have so far not shown any significant interactions, drug interactions should be given appropriate attention when other combinations are used.

Proposed reductive metabolism of artemisinin by glutathione transferases in vitro

Artemisinin is a sesquiterpene lactone containing an endoperoxide bridge. It is a promising new antimalarial and is particularly useful against the drug resistant strains of Plasmodium falciparum. It has unique antimalarial properties since it acts through the generation of free radicals that alkylate parasite proteins. Since the antimalarial action of the drug is antagonised by glutathione and ascorbate and has unusual pharmacokinetic properties in humans, we have investigated if the drug is broken down by a typical reductive reaction in the presence of glutathione transferases. Cytosolic glutathione transferases (GSTs) detoxify electrophilic xenobiotics by catalysing the formation of glutathione (GSH) conjugates and exhibit glutathione peroxidase activity towards hydroperoxides. Artemisinin was incubated with glutathione, NADPH and glutathione reductase and GSTs in a coupled assay system analogous to the standard assay scheme with cumene hydroperoxide as a substrate of GSTs. Artemisinin was shown to stimulate NADPH oxidation in cytosols from rat liver, kidney, intestines and in affinity purified preparations of GSTs from rat liver. Using human recombinant GSTs hetelorogously expressed in Escherichia coli, artemisinin was similarly shown to stimulate NADPH oxidation with the highest activity observed with GST M1-1. Using recombinant GSTs the activity of GSTs with artemisinin was at least two fold higher than the reaction with CDNB. Considering these results, it is possible that GSTs may contribute to the metabolism of artemisinin in the presence of NADPH and GSSG-reductase We propose a model, based on the known reactions of GSTs and sesquiterpenes, in which (1) artemisinin reacts with GSH resulting in oxidised glutathione; (2) the oxidised glutathione is then converted to reduced glutathione via glutathione reductase; and (3) the latter reaction may then result in the depletion of NADPH via GSSG-reductase. The ability of artemisinin to react with GSH in the presence of GST may be responsible for the NADPH utilisation observed in vitro and suggests that cytosolic GSTs are likely to be contributing to metabolism of artemisinin and related drugs in vivo.

Identification of the human cytochrome P450 enzymes involved in the in vitro metabolism of artemisinin.

The study aimed to identify the specific human cytochrome P450 (CYP450) enzymes involved in the metabolism of artemisinin. Microsomes from human B-lymphoblastoid cell lines transformed with individual CYP450 cDNAs were investigated for their capacity to metabolize artemisinin. The effect on artemisinin metabolism in human liver microsomes by chemical inhibitors selective for individual forms of CYP450 was investigated. The relative contribution of individual CYP450 isoenzymes to artemisinin metabolism in human liver microsomes was evaluated with a tree-based regression model of artemisinin disappearance rate and specific CYP450 activities. The involvement of CYP2B6 in artemisinin metabolism was demonstrated by metabolism of artemisinin by recombinant CYP2B6, inhibition of artemisinin disappearance in human liver microsomes by orphenadrine (76%) and primary inclusion of CYP2B6 in the tree-based regression model. Recombinant CYP3A4 was catalytically competent in metabolizing artemisinin, although the rate was 10% of that for recombinant CYP2B6. The tree-based regression model suggested CYP3A4 to be of importance in individuals with low CYP2B6 expression. Even though ketoconazole inhibited artemisinin metabolism in human liver microsomes by 46%, incubation with ketoconazole together with orphenadrine did not increase the inhibition of artemisinin metabolism compared to orphenadrine alone. Troleandomycin failed to inhibit artemisinin metabolism. The rate of artemisinin metabolism in recombinant CYP2A6 was 15% of that for recombinant CYP2B6. The inhibition of artemisinin metabolism in human liver microsomes by 8-methoxypsoralen (a CYP2A6 inhibitor) was 82% but CYP2A6 activity was not included in the regression tree. Artemisinin metabolism in human liver microsomes is mediated primarily by CYP2B6 with probable secondary contribution of CYP3A4 in individuals with low CYP2B6 expression. The contribution of CYP2A6 to artemisinin metabolism is likely of minor importance.

Quantitative in vivo and in vitro sex differences in artemisinin metabolism in rat

1. The pharmacokinetics of the antimalarial compound artemisinin were compared in the male and female Sprague-Dawley rat after single dose i.v. (20 mg.kg) or i.p. (50 mg.kg) administration of an emulsion formulation. 2. Plasma clearance of artemisinin was 12.0 (95% confidence interval: 10.4, 13.0) l.h. kg in the male rat and 10.6 (95% CI: 7.5, 15.0) l.h. kg in the female rat suggesting high hepatic extraction in combination with erythrocyte uptake or clearance. Artemisinin half-life was 0.5 h after both routes of administration in both sexes. Values for plasma clearance and half-lives did not statistically differ between the sexes. 3. After i.p. administration artemisinin AUCs were 2-fold higher in the female compared with male rat (p 0.001). Artemisinin disappearance was 3.9-fold greater in microsomes from male compared with female livers and it was inhibited in male microsomes by goat or rabbit serum containing antibodies against CYP2C11 and CYP3A2 but not CYP2B1 or CYP2E1. 4. The unbound fraction of artemisinin in plasma was lower (p 0.001) in plasma obtained from the male (8.8 2.0%) compared with the female rat (11.7 2.2%). 5. The possibility of a marked sex difference, dependent on the route of administration, has to be taken into account in the design and interpretation of toxicological studies of artemisinin in this species.