Artemisinin

Artemisinin, also known as Qinghaosu (Chinese: 青蒿素) , and its derivatives are a group of drugs that possess the most rapid action of all current drugs against  falciparum malaria. Treatments containing an artemisinin derivative (artemisinin-combination therapies, ACTs) are now standard treatment worldwide for falciparum malaria. The starting compound artemisinin is isolated from the plant Artemisia annua, a herb described in Chinese traditional medicine.

Chemically, artemisinin is a sesquiterpene lactone containing an unusual peroxide bridge. It is believed that this peroxide is responsible for the drug's mechanism of action. No other natural compound with such a peroxide bridge is known.

Use of the drug by itself as a monotherapy is explicitly discouraged by the World Health Organization as there have been signs that malarial parasites are developing resistance to the drug. Therapies that combine artemisinin with some other anti-malarial drug are the preferred treatment for malaria and are both effective and well tolerated in patients. The drug is also increasingly being used in vivax malaria as well as being a topic of research in cancer treatment.

History
Artemisia has been used by Chinese herbalists for more than a thousand years in the treatment of many illnesses, such as skin diseases and malaria. The earliest record dates back to 200 BC, in the "Fifty-two Prescriptions" unearthed from the Mawangdui Han Dynasty Tombs. Its antimalarial application was first described, in Zhouhou Beiji Fang ("The Handbook of Prescriptions for Emergencies", ), edited in the middle of the fourth century by Ge Hong. In that book, 43 malaria treatment methods were recorded, including to shout loudly with a cock in hands, or to eat two halves of one soy bean with Chinese characters for "sun" and "moon" written on.

In the 1960s a research program, under the name Project 523, was set up by the Chinese army to find an adequate treatment for malaria. In 1972, in the course of this research, Dr. Tu Youyou discovered artemisinin in the leaves of Artemisia annua (annual wormwood). The drug is named Qinghaosu in Chinese. It was one of many candidates then tested by Chinese scientists from a list of nearly 5000 traditional Chinese medicines for treating malaria. It was the only one that was effective, but it was found that it cleared malaria parasites from their bodies faster than any other drug in history. Artemisia annua is a common herb and has been found in many parts of the world, including along the Potomac River, in Washington, D.C. Images of the original scientific papers are available online and a book, Zhang Jianfang, "Late Report – Record of Project 523 and the Research and Development of Qinghaosu", Yangcheng Evening News Publisher 2007(張劍方. 遲到的報告五二三項目與青蒿素研發紀實. 羊城晚報出版社, 2007), was published in 2006, which records the history of the discovery.

It remained largely unknown to the rest of the world for about seven years, until results were published in the Chinese Medical Journal in 1979. The report was met with skepticism at first, partly because the chemical structure of artemisinin, particularly the peroxide, appeared to be too unstable to be a viable drug.

For many years after the discovery, access to the purified drug and the plant it was extracted from were restricted by the Chinese government. It was not until the late 1970s and early 80s that news of the discovery reached scientists outside China. The World Health Organisation (WHO) tried to contact Chinese scientists and officials to find out more, but drew a blank. Dr Ying Lee, one of the scientists involved in the research into artemisinin, said the Chinese distrusted the West. The Chinese suspected the West just wanted to exploit the drug and sell it around the world slightly altered and repatented. The fact that there were several Americans on the WHO's steering board on malaria and that some were from the military did not help clear the distrust. It can be noted Americans had just invested a lot into mefloquine, an analogue of quinine.

In 2006, after artemisinin had become the treatment of choice for malaria, the WHO called for an immediate halt to single-drug artemisinin preparations in favor of medications that combine artemisinin with another malaria drug, in order to reduce the risk of parasites developing resistance. In 2011, Dr. Tu was awarded the prestigious Lasker-DeBakey Clinical Medical Research Award for her discovery.

Artemisinin derivatives
Because artemisinin itself has physical properties such as poor bioavailability that limit its effectiveness, semi-synthetic derivatives of artemisinin have been developed. These include:


 * Artesunate (water-soluble: for oral, rectal, intramuscular, or intravenous use)
 * Artemether (lipid-soluble: for oral, rectal or intramuscular use)
 * Dihydroartemisinin
 * Artelinic acid
 * Artenimol
 * Artemotil

There are also simplified analogs in preclinical research.

A synthetic compound with a similar trioxolane structure (ring containing three oxygen atoms) with the name OZ-277, also known as RBx11160 or arterolane, showed promise in in vitro testing. However in phase II testing (in patients with malaria) the drug did not prove as effective as hoped. A combination with piperaquine remains in development.

Uncomplicated Malaria
Artemisinins can be used alone, but this leads to a high rate of recrudescence (return of parasites) and other drugs are required to clear the body of all parasites and prevent recurrence. The World Health Organization is pressuring manufacturers to stop making the uncompounded drug available to the medical community at large, aware of the catastrophe that would result if the malaria parasite developed resistance to artemisinins.

The World Health Organisation has recommended that artemisinin combination therapies (ACT) be first-line therapy for P. falciparum malaria worldwide. Combinations are effective because the artemisinin component kills the majority of parasites at the start of the treatment while the more slowly eliminated partner drug clears the remaining parasites.

Several fixed-dose ACTs are now available containing an artemisinin component and a partner drug which has a long half-life, such as mefloquine (ASMQ ), lumefantrine (Coartem), amodiaquine (ASAQ), piperaquine (Duo-Cotecxin) and pyronaridine (Pyramax). Increasingly these combinations are being made to GMP standard. A separate issue concerns the quality of some artemisinin-containing products being sold in Africa and South-East Asia.

Artemisinins are not used for malaria prophylaxis (prevention) because of the extremely short activity (half-life) of the drug. To be effective, it would have to be administered multiple times each day.

Severe Malaria
Artesunate administered by intravenous or intramuscular injection has proven superior to quinine in large, randomised controlled trials in both adults and children. Combining all trials comparing these two drugs, artesunate is associated with a mortality rate that is approximately 30% lower than that of quinine. Reasons for this difference include reduced incidence of hypoglycaemia, easier administration and more rapid action against circulating and sequestered parasites.

Cancer treatment
Artemisinin is undergoing early research and testing for the treatment of cancer, primarily by researchers at the University of Washington. Chinese scientists have shown artemisinin has significant anticancer effects against human hepatoma cells. Artemisinin has a peroxide lactone group in its structure, and it is thought that when the peroxide comes into contact with high iron concentrations (common in cancerous cells), the molecule becomes unstable and releases reactive oxygen species. It has been shown to reduce angiogenesis and the expression of vascular endothelial growth factor in some tissue cultures. Recent pharmacological evidence demonstrates that the artemisinin-derivative dihydroartemisinin targets human metastatic melanoma cells with induction of NOXA (Phorbol-12-myristate-13-acetate-induced protein 1)-dependent mitochondrial apoptosis that occurs downstream of iron-dependent generation of cytotoxic oxidative stress.

Helminth parasites
Serendipitous discovery was made in China while searching for novel anthelmintics for schistosomiasis. Artemisinin was effective against schistosomes, the human blood flukes, which are the second most prevalent parasitic infections, after malaria. Artemisinin and its derivatives are all potent anthelmintics. Artemisinins were later found to possess a broad spectrum of activity against a wide range of trematodes including Schistosoma japonicum, S. mansoni, S. haematobium, Clonorchis sinensis, Fasciola hepatica and Opisthorchis viverrini. Clinical trials were also successfully conducted in Africa among patients with schistosomiasis. A randomized, double-blind placebo-controlled trial also revealed the efficacy against schistosome infection in Côte d'Ivoire and China.

Resistance
A 2008 study suggests a consensus among researchers that artemisinin is losing its potency in Cambodia and increased efforts are required to prevent drug-resistant malaria from spreading across the globe. These findings were subsequently supported by a detailed study from Western Cambodia.

In April 2011 the World Health Organization stated that resistance to the most effective anti-malarial drug, artemisinin, could unravel national (India) malaria control programs, which have achieved significant progress in the last decade. WHO advocates the rational use of anti-malarial drugs and acknowledges the crucial role of community health workers in reducing malaria in the region.

Adverse effects
Artemisinins are generally well tolerated at the doses used to treat malaria. The side effects from the artemisinin class of medications are similar to the symptoms of malaria: nausea, vomiting, anorexia, and dizziness. Mild blood abnormalities have also been noted. A rare but serious adverse effect is allergic reaction. One case of significant liver inflammation has been reported in association with prolonged use of a relatively high-dose of artemisinin for an unclear reason (the patient did not have malaria). The drugs that are used in combination therapies can contribute to the adverse effects that are experienced by those undergoing treatment. Adverse effects in patients with acute falciparum malaria treated with artemisinin derivatives tend to be higher.

Mechanism of action
There is no consensus regarding the mechanism through which artemisinin derivatives kill the parasites. Their site of action within the parasite also remains controversial.

All artemisinins used today are prodrugs of the biologically active metabolite dihydroartemisinin, which is active during the stage when the parasite is located inside red blood cells.

When the parasite that causes malaria infects a red blood cell, it consumes hemoglobin within its digestive vacuole, liberating free heme, an iron-porphyrin complex. According to one theory about the action of artemisinin, the iron of the heme reduces the peroxide bond in artemisinin, generating high-valent iron-oxo species and resulting in a cascade of reactions that produce reactive oxygen radicals which damage the parasite and lead to its death.

Numerous studies have investigated the type of damage that oxygen radicals may induce. For example, Pandey et al. have observed inhibition of digestive vacuole cysteine protease activity of malarial parasite by artemisinin. These observations were supported by ex vivo experiments showing accumulation of hemoglobin in the parasites treated with artemisinin and inhibition of hemozoin formation by malaria parasites. Electron microscopic evidence linking artemisinin action to the parasite's digestive vacuole has been obtained showing that the digestive vacuole membrane suffers damage soon after parasites are exposed to artemisinin. This would also be consistent with data showing that the digestive vacuole is already established by the mid-ring stage of the parasite's blood cycle, a stage that is sensitive to artemisinins but not other antimalarials.

A commonly cited theory that the parasite's SERCA pump (PfATP6) is a key target of artemisinins now appears highly questionable. Artemisinins do not inhibit this protein when it is expressed in yeast and transfection studies show no significant effect of PfATP6 genotype on artemisinin phenotype. Although a single field study identified a mutation in PfATP6 that was associated with resistance to artemether, this mutation has not achieved a meaningful prevalence in any location and the phenotypic association is not replicated elsewhere. There is no role for PfSERCA in the artemisinin resistance that appears to be emerging in Cambodia.

A 2005 study investigating the mode of action of artemisinin using a yeast model demonstrated that the drug acts on the electron transport chain, generates local reactive oxygen species, and causes the depolarization of the mitochondrial membrane.

Dosing
Artemisinin and its derivatives have half-lives in the order of a few hours and therefore require at least daily dosing, usually for three days. For example the WHO approved adult dose of co-artemether (artemether-lumefantrine) is 4 tablets at 0, 8, 24, 36, 48 and 60 hours (six doses). This regimen has been proven to be superior to regimens based on amodiaquine.

Artemisinin is not soluble in water and therefore Artemisia annua tea was postulated not to contain pharmacologically significant amounts of artemesinin. However, this conclusion was rebuked by several experts who stated that hot water (85 oC), and not boiling water, should be used to prepare the tea. Although Artemisia tea is not recommended as a substitute for the ACT (artemisinin combination therapies) more clinical studies on artemisia tea preparation have been suggested.

Production and price
Artemisinin is the only high-volume drug that continues to be produced from a plant-based source. China and Vietnam provide 70% and East Africa 20% of the raw material. Seedlings are grown in nurseries and then transplanted into fields. It takes about 8 months for them to reach full size. The plants are harvested, the leaves are dried and sent to facilities where the artemisinin is extracted using solvent, typically hexane. The market price for artemisinin has fluctuated widely, between $120 and $1200 per kilogram from 2005 to 2008.

After negotiation with the WHO, Novartis and Sanofi-Aventis provide ACT (artemisinin combination therapy) drugs at cost on a non-profit basis; however, these drugs are still more expensive than other malaria treatments.

High-yield varieties of Artemisia are being produced by the Centre for Novel Agricultural Products at the University of York using molecular breeding techniques.

Using seed supplied by Action for Natural Medicine (ANAMED), the World Agroforestry Centre (ICRAF) has developed a hybrid, dubbed A3, which can grow to a height of 3 m and which produces 20 times more artemisinin than wild varieties. In northwestern Mozambique, ICRAF is working together with a medical organisation, Médecins sans frontières, ANAMED and the Ministry of Agriculture and Rural Development to train farmers on how to grow the shrub from cuttings, and to harvest and dry the leaves to make artemisia tea.

A research group from the Philippines have published a work on the extraction of artemisinin from its plant source, Artemisia annua. They produced a synthetic polymer that was imprinted with artemisinin and showed a selective recognition to said compound.

Biosynthesis in A. annua
The biosynthesis of artemisinin is expected to involve the mevalonate pathway (MVA) and the cyclization of FDP (farnesyl diphosphate). Although it is not clear whether the DXP (deoxyxylulose phosphate)pathway can also contribute 5-carbon precursors (IPP or/and DMAPP), as occurs in other sesquiterpene biosynthetic systems. The routes from artemisinic alcohol to artemisinin remain controversial and they differ mainly in when the reduction step takes place. Both routes suggested dihydroartemisinic acid as the final precursor to artemisinin. Dihydroartemisinic acid then undergoes photo-oxidation to produce dihydroartemisinic acid hydroperoxide. Ring expansion by the cleavage of hydroperoxide and a second oxygen-mediated hydroperoxidation furnish the biosynthesis of artemisinin.



Figure 1. Biosynthesis of Artemisinin.

Chemical synthesis
The total synthesis of artemisinin, while expensive, can be performed using basic organic reagents. In 1982, G. Schmid and W. Hofheinz published a paper showing the complete synthesis of artemisinin. Their starting material was (-)-Isopulegol (2) which as converted to methoxymethyl ether (3). The ether was hydroborated and then underwent oxidative workup to give (4). The primary hydroxyl group was then benzylated and the methoxymethyl ether was cleaved resulting in (5) which would be oxidized to (6). Next, the compound was protonated and treated with (E)-(3-iodo-1-methyl-1-propenyl)-trimethylsilane to give (7). This resulting ketone was reacted with lithium methoxy(trimethylsily)methylide to obtain two diastereomeric alcohols, (8a) and (8b). 8a was then debenzylated using (Li, ) to give lactone (9). The vinylsilane was then oxidized to ketone (10). The ketone was then reacted with fluoride ion that caused it to undergo desilylation, enol ether formation and carboxylic acid formation to give (11). An introduction of a hydroperoxide function at C(3) of 11 gives rise to (12). Finally, this underwent photooxygenation and then treated with acid to produce artemisinin.

Synthesis in engineered organisms
In 2006 a team from UC Berkeley reported that they had engineered Saccharomyces cerevisiae yeast to produce the precursor artemisinic acid. The synthesized artemisinic acid can then be transported out, purified and chemically converted into artemisinin that they claim will cost roughly 0.25 cents per dose. In this effort of synthetic biology, a modified mevalonate pathway was used and the yeast were engineered to express the enzyme amorphadiene synthase and a cytochrome P450 monooxygenase (CYP71AV1), both from A. annua. A three-step oxidation of amorpha-4,11-diene gives the resulting artemisinic acid. Amyris Inc. collaborated with UC Berkeley and the Institute for One World Health to further develop this technology. The collaboration, known as the Artemisinin Project, is supported by funding from the Bill & Melinda Gates Foundation, and aims to create a source of non-seasonal, high-quality and affordable artemisinin to supplement the botanical supply, with the objective of making ACTs more accessible. The technology is based on inventions licensed from UC Berkeley and the National Research Council (NRC) Plant Biotechnology Institute of Canada. In 2011, OneWorld Health announced that the project has entered the "production and distribution" phase. Integration of semisynthetic artemisinin into the supply chain is planned for 2012.

According to the WHO World Malaria Report 2010, artemisinin from yeast will not become available on the market until at least 2012 and will only cover part of the global requirement, which is where semisynthetic artemisinin will make a remarkable difference by alleviating shortages and ensuring that malaria treatment is available to the patients worldwide.

In 2010 a team from Wageningen University reported that they had engineered a close relative of tobacco, Nicotiana benthamiana, that can also produce the precursor artemisinic acid.