Making artemisinin

The possibilities for the production of the antimalarial artemisinin by biological and chemical means are explored. These include native biosynthesis, genetic modification of Artemisia annua and other plants, engineering of microbes, total and partial chemical synthesis and combinations of the above.     

Effect of artemisinin on proliferation and apoptosis-related protein expression in vivo and in vitro

Artemisinin is the first-line drugs for the treatment of malaria. In recent years, a large number of reports showed that artemisinin exhibit anti-tumor activity. In this study, we used C6 glioma cells and rat C6 brain-glioma model to study anti-tumor activity of artemisinin in vivo and in vitro. We found that artemisinin inhibited the proliferation in C6 cells and induced cell cycle arrest and a caspase-3-dependent cell apoptosis. It also inhibited the growth of C6 brain-glioma in vivo and enhanced living state of rat brain-glioma model. These results suggested that artemisinin had significant anti-tumor activities on C6 cells both in vitro and in vivo. Artemisinin might be exploited as a promising clinical anti-cancer drug in future.     

Production of artemisinin by genetically-modified microbes

Artemisinin, an endoperoxidized sesquiterpene originally extracted from the medicinal plant Artemisia annua L., is a potent malaria-killing agent. Due to the urgent demand and short supply of this new antimalarial drug, engineering enhanced production of artemisinin by genetically-modified or transgenic microbes is currently being explored. Cloning and expression of the artemisinin biosynthetic genes in Saccharomyces cerevisiae and Escherichia coli have led to large-scale microbial production of the artemisinin precursors such as amorpha-4,11-diene and artemisinic acid. Although reconstruction of the complete biosynthetic pathway toward artemisinin in transgenic yeast and bacteria has not been achieved, artemisinic acid available from these transgenic microbes facilitates the subsequent partial synthesis of artemisinin by either chemical or biotransformational process, thereby providing an attractive strategy alternative to the direct extraction of artemisinin from A.annua L. In this review, we update the current trends and summarize the future prospects on genetic engineering of the microorganisms capable of accumulating artemisinin precursors through heterologous and functional expression of the artemisinin biosynthetic genes.     

Enhanced artemisinin production from engineered yeast precursors upon biotransformation

Abstract

Production of artemisinin in genetically modified microorganisms is an attractive option to enable sufficient supply of the effective antimalarial agent. Although a sundry of artemisinin precursors are available from engineered bacteria or yeast, no artemisinin has been manufactured by engineering any microbial platforms due to inaccessibility to unidentified steps. To this end, it is essential to consider how to convert artemisinin precursors to artemisinin, either biochemically or chemically. To establish a novel procedure of artemisinin production, we incubate the mixture of artemisinin precursors from engineered Sacchromyces cerevisiae with the cell-free enzyme extract of Artemisia annua. For the single gene-expressing strain INVScI (pYES-ADS), amorpha-4,11-diene accumulation within 48 h or 14 days led to higher artemisinin content than the control. In the multiple gene-expressing strain YPH501 (pYES-ADS:: pESC-CYP71AV1-DBR2), artemisinin accumulation from the 14-day-induced yeast precursor mixture was nearly equivalent between the single gene-transferred strain and the multiple gene-transferred strain. Alternatively, biotransformation of 48-hour-induced yeast amorpha-4,11-diene mixture by the cold-acclimated A. annua cell-free extract that possesses the abundant enzymes relevant to artemisinin biosynthesis gave rise to considerable elevation of artemisinin content up to 0.647% in maximum, accounting to 15-folds increase as the A. annua cell-free extract without cold-acclimation (0.045%), thereby providing a practical protocol for artemisinin overproduction through the interplay of engineered microbial artemisinin precursors with upregulated plant enzymes.     

Sugars proportionately affect artemisinin production

Little is known about the effect of sugars in controlling secondary metabolism. In this study, sugars alone or in combination with their analogs were used to investigate their role in the production of the antimalarial drug, artemisinin, in Artemisia annua L. seedlings. Compared to sucrose, a 200% increase in artemisinin by glucose was observed. Different ratios of fructose to glucose yielded artemisinin levels directly proportional to increases in relative glucose concentration. When the glucose analog, 3-O-methylglucose, was added with glucose, artemisinin production was dramatically decreased, but hexokinase activity was significantly increased compared to glucose alone. In contrast, neither mannose nor mannitol had any significant effect on artemisinin yield. In comparison with 30 g/l sucrose, artemisinin levels were significantly reduced by 80% in the presence of 27 g/l sucrose + 3 g/l palatinose, which cannot be transported into cells through the sucrose transporter. Together these results suggest that both monosaccharide and disaccharide sugars are likely acting not only as carbon sources but also as signals to affect the downstream production of artemisinin, and that the mechanism of these effects appears to be complex.     

Synthesis of artemisinin dimers using the Ugi reaction and their in vitro efficacy on breast cancer cells

The Ugi four-component reaction was used to prepare a series of artemisinin monomers and dimers. We found that the endoperoxide group in artemisinin remains intact during the reaction. The new artemisinin dimers showed potent anti-cancer activity against two human breast cancer cell lines, MDA-MB-231 and BT-474. One of the Ugi artemisinin dimers showed an IC₅₀ value of 12 nM when tested on BT474 cells, more than 600 times more potent than artesunate. Furthermore, the same Ugi artemisinin dimer showed a low toxicity when tested on MCF10A, a nontumorigenic cell line, resulting in a selectivity index of more than 8000.     

Transgenic approach to increase artemisinin content in Artemisia annua L.

Artemisinin, the endoperoxide sesquiterpene lactone, is an effective antimalarial drug isolated from the Chinese medicinal plant Artemisia annua L. Due to its effectiveness against multi-drug-resistant cerebral malaria, it becomes the essential components of the artemisinin-based combination therapies which are recommended by the World Health Organization as the preferred choice for malaria tropica treatments. To date, plant A. annua is still the main commercial source of artemisinin. Although semi-synthesis of artemisinin via artemisinic acid in yeast is feasible at present, another promising approach to reduce the price of artemisinin is using plant metabolic engineering to obtain a higher content of artemisinin in transgenic plants. In the past years, an Agrobacterium-mediated transformation system of A. annua has been established by which a number of genes related to artemisinin biosynthesis have been successfully transferred into A. annua plants. In this review, the progress on increasing artemisinin content in A. annua by transgenic approach and its future prospect are summarized and discussed.     

Amorpha-4,11-diene synthase: cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin

The sesquiterpenoid artemisinin, isolated from the plant Artemisia annua L., and its semi-synthetic derivatives are a new and very effective group of antimalarial drugs. A branch point in the biosynthesis of this compound is the cyclisation of the ubiquitous precursor farnesyl diphosphate into the first specific precursor of artemisinin, namely amorpha-4,11-diene. Here we describe the isolation of a cDNA clone encoding amorpha-4,11-diene synthase. The deduced amino acid sequence exhibits the highest identity (50%) with a putative sesquiterpene cyclase of A. annua. When expressed in Escherichia coli, the recombinant enzyme catalyses the formation of amorpha-4,11-diene from farnesyl diphosphate. Introduction of the gene into tobacco (Nicotiana tabacum L.) resulted in the expression of an active enzyme and the accumulation of amorpha-4,11-diene ranging from 0.2 to 1.7 ng per g fresh weight. Received: 8 June 2000 / Accepted: 21 August 2000     

Bio-transformation of artemisinin using soil microbe: Direct C-acetoxylation of artemisinin at C-9 by Penicillium simplissimum

Potent antimalarial compound artemisinin, 1 was bio-transformed to C-9 acetoxy artemisinin, 2 using soil microbe Penicillium simplissimum along with C-9 hydroxy derivative 3. The products were characterized using high field NMR and MS–MS data. The absolute stereochemistry of the newly generated chiral centers has been ascertained by COSY and 1D NOESY experiments. This is the first Letter of direct C-acetoxylation of artemisinin using microbial strains.     

Cytotoxic activity of secondary metabolites derived from Artemisia annua L. towards cancer cells in comparison to its designated active constituent artemisinin

Artemisia annua L. (sweet wormwood, qinhao) has traditionally been used in Chinese medicine. The isolation of artemisinin from Artemisia annua and its worldwide accepted application in malaria therapy is one of the showcase success stories of phytomedicine during the past decades. Artemisinin-type compounds are also active towards other protozoal or viral diseases as well as cancer cells in vitro and in vivo. Nowadays, Artemisia annua tea is used as a self-reliant treatment in developing countries. The unsupervised use of Artemisia annua tea has been criticized to foster the development of artemisinin resistance in malaria and cancer due to insufficient artemisinin amounts in the plant as compared to standardized tablets with isolated artemisinin or semisynthetic artemisinin derivatives. However, artemisinin is not the only bioactive compound in Artemisia annua. In the present investigation, we analyzed different Artemisia annua extracts. Dichloromethane extracts were more cytotoxic (range of IC₅₀: 1.8-14.4 μg/ml) than methanol extracts towards Trypanosoma b. brucei (TC221 cells). The range of IC₅₀ values for HeLa cancer cells was 54.1-275.5 μg/ml for dichloromethane extracts and 276.3-1540.8 μg/ml for methanol extracts. Cancer and trypanosomal cells did not reveal cross-resistance among other compounds of Artemisia annua, namely the artemisinin-related artemisitene and arteanuine B as well as the unrelated compounds, scopoletin and 1,8-cineole. This indicates that cells resistant to one compound retained sensitivity to another one. These results were also supported by microarray-based mRNA expression profiling showing that molecular determinants of sensitivity and resistance were different between artemisinin and the other phytochemicals investigated.     

Evidence of artemisinin production from IPP stemming from both the mevalonate and the nonmevalonate pathways

The potent antimalarial sesquiterpene lactone, artemisinin, is produced in low quantities by the plant Artemisia annua L. The source and regulation of the isopentenyl diphosphate (IPP) used in the biosynthesis of artemisinin has not been completely characterized. Terpenoid biosynthesis occurs in plants via two IPP-generating pathways: the mevalonate pathway in the cytosol, and the non-mevalonate pathway in plastids. Using inhibitors specific to each pathway, it is possible to resolve which supplies the IPP precursor to the end product. Here, we show the effects of inhibition on the two pathways leading to IPP for artemisinin production in plants. We grew young (7–14 days post cotyledon) plants in liquid culture, and added mevinolin to the medium to inhibit the mevalonate pathway, or fosmidomycin to inhibit the non-mevalonate pathway. Artemisinin levels were measured after 7–14 days incubation, and production was significantly reduced by each inhibitor compared to controls, thus, it appears that IPP from both pathways is used in artemisinin production. Also when grown in miconazole, an inhibitor of sterol biosynthesis, there was a significant increase in artemisinin compared to controls suggesting that carbon was shifted from sterols into sesquiterpenes. Collectively these results indicate that artemisinin is probably biosynthesized from IPP pools from both the plastid and the cytosol, and that carbon from competing pathways can be channeled toward sesquiterpenes. This information will help advance our understanding of the regulation of in planta production of artemisinin.     

Hemin potentiates the anti-hepatitis C virus activity of the antimalarial drug artemisinin

We report that the antimalarial drug artemisinin inhibits hepatitis C virus (HCV) replicon replication in a dose-dependent manner in two replicon constructs at concentrations that have no effect on the proliferation of the exponentially growing host cells. The 50% effective concentration (EC(50)) for inhibition of HCV subgenomic replicon replication in Huh 5-2 cells (luciferase assay) by artemisinin was 78+/-21 microM. Hemin, an iron donor, was recently reported to inhibit HCV replicon replication [mediated by inhibition of the viral polymerase (C. Fillebeen, A.M. Rivas-Estilla, M. Bisaillon, P. Ponka, M. Muckenthaler, M.W. Hentze, A.E. Koromilas, K. Pantopoulos, Iron inactivates the RNA polymerase NS5B and suppresses subgenomic replication of hepatitis C virus, J. Biol. Chem. 280 (2005) 9049-9057.)] at a concentration that had no adverse effect on the host cells. When combined, artemisinin and hemin resulted, over a broad concentration range, in a pronounced synergistic antiviral activity. Also at a concentration (2 microM) that alone had no effect on HCV replication, hemin still potentiated the anti-HCV activity of artemisinin.     

The reaction of artemisinin with hemin: a further insight into the mechanism

The mechanism of action of trioxane antimalarial drugs is still largely controversial and warrants further investigation. We report here on the direct reaction of artemisinin with hemin, carried out in DMSO, in the absence of reducing agents. The reaction was analysed, independently, by visible spectroscopy, HPLC-ESI/MS and ¹H NMR. Two isomeric artemisinin-hemin long-lived adducts are unambiguously detected. Eventual degradation of the porphyrin ring and loss of the Soret band are observed as well. Implications of the present results for the mechanism of action of artemisinin-based antimalarials are discussed.     

Combination of zingerone and dihydroartemisinin presented synergistic antimalarial activity against Plasmodium berghei infection in BALB/c mice as in vivo model

Malaria is a global health problem leading to the death of 435,000 cases in tropical and sub-tropical zones. Spread and emergence of increasing resistance to the antimalarial drugs are the major challenges in the control of malaria. Therefore, searching for alternative antimalarial drugs is urgently needed, and combination treatment preferred as an approach to address this. This study aimed to evaluate in vivo antimalarial activity of zingerone (ZN), and its combination with dihydroartemisinin (DHA) against Plasmodium berghei infected mice. ZN was prepared and tested for acute oral toxicity according to the OECD guideline. In vivo antimalarial activity of different doses of ZN and combination with DHA were determined using the 4-day suppression test. The results showed that ZN was found to be safe and no mortality within the observation period, and the lethal dose might be greater than the limited dose of 1000 mg/kg. For in vivo antimalarial test, ZN exhibited significant (p < .05) parasitemia inhibition of 30.65% and 45.75% at the doses of 50 mg/kg and 100 mg/kg, respectively. Moreover, effective dose 50 (ED₅₀) of ZN was 29.76 mg/kg. The combination treatment of ZN and DHA at the doses of ED₅₀ values at the fixed ratio 1:1 was found to present significant (p < .001) antimalarial activity as compared to ZN and DHA treated alone with markedly prolonged mean survival time. Additionally, the combination index (0.83384) revealed the synergistic antimalarial effect. It can be concluded that ZN exerted potent antimalarial activity with no toxicity, and combination treatment with DHA produced the synergistic antimalarial effect.     

Microbial transformation of antimalarial terpenoids

The fungal and bacterial transformation of terpenoids derived from plant essential oils, especially the sesquiterpenoid artemisinin from Artemisia annua, has produced several new candidate drugs for the treatment of malaria. Obtaining new derivatives of terpenoids, including artemisinin derivatives with increased antimalarial activity, is an important goal of research in microbial biotechnology and medicinal chemistry.     

On the possible role of qinghao acid in the biosynthesis of artemisinin

Artemisinin (qinghaosu), a seco-sesquiterpene peroxide, is the clinically established antimalarial principle isolated from the leaves of the Chinese medicinal herb, Artemisia annua. Recent studies have suggested that arteannuin B, another metabolite of this plant, could serve as a precursor for artemisinin. In the present study, qinghao acid, the major sesquiterpene constituent of A. annua, was converted to arteannuin B by singlet oxygen (¹O₂) generated by sensitized photo-oxygenation. The formation of this compound was monitored by high-pressure liquid chromatographic analysis, and the identity of the isolated material was established by direct comparison. Since ¹O₂ is known to play a role in biogenetic reactions, it appears that qinghao acid can serve as a biogenetic precursor for artemisinin.     

Combined antimalarial therapy using artemisinin

The existing armamentarium of drugs for the treatment and prevention of malaria is limited primarily by resistance (and cross-resistance between closely related drugs). However, most of these drugs still have a place and their life-span could be prolonged if better deployed and used, and also by rationally combining them based on pharmacodynamic and pharmacokinetic properties. Newer compounds are also being developed. The nature of malaria disease and its prevalence in the developing world call for innovative approaches to develop new affordable drugs and to safeguard the available ones. According to WHO, the concept of combination therapy is based on the synergistic or additive potential of two or more drugs, to improve therapeutic efficacy and also delay the development of resistance to the individual components of the combination. Combination therapy (CT) with antimalarial drugs is the simultaneous use of two or more blood schizontocidal drugs with independent modes of action and different biochemical targets in the parasite. In the context of this definition, multiple-drug therapies that include a nonantimalarial drug to enhance the antimalarial effect of a blood schizontocidal drug are not considered combination therapy. Similarly, certain antimalarial drugs that fit the criteria of synergistic fixed-dose combinations are operationally considered as single products in that neither of the individual components would be given alone for anti-malarial therapy. An example is sulfadoxine-pyrimethamine. Artemisinin-based combination therapies have been shown to improve treatment efficacy and also contain drug resistance in South-East Asia. However, major challenges exist in the deployment and use of antimalarial drug combination therapies, particularly in Africa. These include: 1) the choice of drug combinations best suited for the different epidemiological situations; 2) the cost of combination therapy; 3) the timing of the introduction of combination therapy; 4) the operational obstacles to implementation, especially compliance. As a response to increasing levels of antimalarial resistance, the World Health Organization (WHO) recommends that all countries experiencing resistance to conventional monotherapies, such as chloroquine, amodiaquine or sulfadoxine/pyrimethamine, should use combination therapies, preferably those containing artemisinin derivatives (ACTs--artemisinin-based combination therapies) for malaria caused by Plasmodium falciparum. There is a promising role of such compounds in replacing or complementing current options. Since 1979, several different formulations of artemisinin and its derivatives have been produced and studied in China in several thousand patients for either P. falciparum or P. vivax malaria. To date, there is no evidence of drug resistance to these compounds. The use of artemisinin, artemether, arteether and artesunate for either uncomplicated or severe malaria is now spreading through almost all malarious areas of the world, although some of they have no patent protection, their development (with few exceptions) has not followed yet full international standards. Both artesunate, artemether and arteether are rapidly and extensively converted to their common bioactive metabolite, dihydroarte-misinin. WHO currently recommends the following therapeutic options: 1) artemether/lumefantrine; 2) artesunate plus amodiaquine; 3) artesunate plus sulfadoxine/pyrimethamine (in areas where SP efficacy remains high); 4) artesunate plus mefloquine (in areas with low to moderate transmission); and 5) amodiaquine plus sulfadoxine/pyrimethamine, in areas where efficacy of both amodiaquine and sulfadoxine/pyrimethamine remains high (mainly limited to countries in West Africa). This non artemisinin-based combination therapy is reserved as an interim option for countries, which, for whatever reason, are unable immediately to move to ACTs.