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New Life for Ancient Malaria Remedy

Credit: Paula Bronstein / iStockphoto

A Burmese boy suffering from malaria is held by his mother at a special clinic for malaria. Credit: Paula Bronstein / iStockphoto

By Nick Klonis & Leann Tilley

The parasite responsible for malaria is developing resistance to a frontline drug that was first used in China more than 2000 years ago. By determining how artemisinin works, scientists may have just opened a new battlefront in the war against malaria.

In the 5th century BC, Hippocrates described a sickness that was caused by breathing the air of fetid swamps. The disease became known as malus aria – Latin for “bad air”.

It was not until just over 100 years ago that protozoan parasites were identified as the causative agent of the disease. These deceptively simple single-celled organisms exhibit a very complex lifecycle involving a human and a mosquito.

Following a bite from an infected female Anopheles mosquito during a “blood meal”, the parasite enters the human bloodstream. Its first port of call is the liver, where it produces thousands of daughter parasites that are re-released into the circulation. Here, the parasites invade red blood cells and undergo further rounds of multiplication, causing fever and sometimes life-threatening illness in the victim. After a period of development, the parasite changes again and can be taken up by a mosquito to continue the cycle.

The World Health Organization currently estimates that one child dies every minute from infection with the most pathogenic human malaria parasite, Plasmodium falciparum. As dreadful as these statistics are, we have seen a 20% decrease in the number of deaths from malaria in the past decade, giving hope that malaria can be beaten.

These improvements are the result of the widespread use of insecticide-treated bed nets and the availability of artemisinin – an “old drug” that has been rediscovered. During China’s Jin Dynasty (265–420 AD), alchemist Hong Ge described a water-brewed extract of Artemisia annua (sweet wormwood) for the treatment of fevers. This was the first reported antimalarial treatment.

Artemisinin became available as a modern drug in the 1970s when a consortium of Chinese scientists, led by Dr Youyou Tu, screened traditional Chinese medicinal plants for antimalarial compounds. After the successful extraction of artemisinin from Artemisia annua and the determination of its structure, artemisinin was introduced to the rest of the world in 1979.

In May 2004, the World Health Organization recommended the use of artemisinin, in combination with other drugs, as the first line treatment for malaria. This year’s Lasker DeBakey Clinical Research Award was awarded to Dr Tu for the discovery of artemisinin.

Artemisinin combination therapy has an efficacy of about 97% in curing P. falciparum malaria, and saves millions of lives every year. Most importantly it acts quickly. Malaria can be a very rapid killer, and there is no use having a drug that takes 48 hours to work if you are likely to die in the next 12 hours.

In other ways artemisinin is not optimal. It lasts for only a short time in the bloodstream and thus it may not be around for long enough to kill all the parasites in the patient’s body. This is one reason why it must always be used in combination with another drug.

Despite its efficacy, we are probably too reliant on this one class of drugs. Almost all the antimalarial drugs in the development pipeline include an artemisinin-related component.

Another cause for worry is that there is early evidence that artemisinin is becoming less potent. In some malarious areas the drug has not been used wisely, and this means that the parasite has been exposed to sub-optimal treatment. This may be responsible for recent reports of delayed parasite clearance times after treatment with artemisinin in some regions of Cambodia.

To lose this drug to resistance would be a major blow to efforts to reduce the malaria burden.

In spite of its widespread use in the field, we are still trying to understand how artemisinin kills malaria parasites. It is critical to understand the mechanism of action so that strategies can be implemented to circumvent artemisinin resistance.

Our research has focused on the blood stage of the parasite lifecycle. This is the part of the lifecycle that is associated with most of the symptoms of malaria and its fatal consequences, and is also the part of the lifecycle that is targeted by most of the available antimalarial drugs, including artemisinin.

The blood stage of the cycle begins when the parasite invades a red blood cell. These cells are essentially “sacks” that are jam-packed with haemoglobin – the protein responsible for transporting oxygen and carbon dioxide around the body.

Most of the mass of haemoglobin is made up of protein – the “globin” part of its name. The “haem” corresponds to a small iron-containing molecule that is intimately associated with globin.

It is the haem of haemoglobin that is responsible for binding oxygen and producing the red colour of blood. Haem is a fascinating molecule: when associated with the globin it is fairly benign, binding oxygen in the lungs and releasing it in the tissues where it is needed.

However, take haem out of the protein and it becomes a very toxic molecule. This is largely due to the presence of iron, which can generate free radicals and cause oxidative damage to the cell.

During its time in the red blood cell, the parasite chews up the haemoglobin to obtain protein-building blocks and also to create space in which it can grow. This leaves the parasite with the problem of how to dispose of the potentially toxic haem.

It does this by converting it into a crystalline compound called haemozoin. This can be easily detected under the microscope as a black pigment in the infected red blood cell. In fact, it was the observation of this parasite “waste product” during microscopic examination of blood smears that led to the identification of the parasite as the causative agent of the disease. Many antimalarial agents work by preventing the detoxification of haem.

So, how does artemisinin work? A number of groups have been examining this question but there is still active debate.

Our studies have shown that the key lies in the fact that the malaria parasite has to consume haemoglobin to survive in red blood cells. We figured that this diet of haemoglobin in the presence of artemisinin might be the parasite’s Achilles’ heel.

Other researchers in the field had argued against a role for hemoglobin digestion in the mechanism of action of artemisinin because artemisinin is active against young parasites, and it had been assumed that hemoglobin digestion didn’t occur until the parasites reached a more mature stage.

However, recent work from our group using high resolution electron microscopy and fluorescence imaging of live cells showed that the digestion process occurs much earlier in the growth cycle than had previously been appreciated.

We designed a number of different strategies to inhibit or slow hemoglobin digestion without compromising the parasite’s viability. We showed that, in the absence of hemoglobin digestion, malaria parasites are essentially resistant to artemisinin.

This revealed clearly that hemoglobin digestion is essential for artemisinin to work. Combined with work from other groups, our experiments tell us that when haemoglobin is broken down it releases iron-containing products that react with artemisinin. This activates the drug to form highly toxic radical species inside the parasite. A downstream effect is oxidative damage to proteins and membranes, eventually overwhelming the parasite’s defence mechanisms.

We have developed experimental methods that allow us to distinguish live from dead parasites following artemisinin treatment. Of particular interest is our observation that parasites that survive artemisinin often have reduced growth compared with untreated parasites.

Our work and that of our colleagues suggests that sub-optimal concentrations of the drug cause the parasite to enter a state of suspended animation that, in Shakespeare’s words, is a “borrowed likeness of shrunk death”: the parasite appears to shut down its metabolism and to stop consuming haemoglobin. This means that artemisinin is not activated, and the parasite survives.

Unfortunately, because artemisinin lasts for only a very short period of time in the bloodstream, even a short period of dormancy is enough to avoid the drug’s toxic mechanism. This may explain the decreased clinical potency of artemisinin that is emerging. Exposure of parasites to sub-optimal regimens may have led to the selection of parasites that have an increased ability to enter a dormant state.

Excitingly our work suggests ways to get around the resistance mechanism. Making artemisinin-like drugs that last for a longer period in the bloodstream would make it much more difficult for the parasite to escape its toxic effects. Such drugs, including one being developed by Prof Sue Charman of Monash University, are currently in pre-clinical trials.

Our work suggests that the new, longer-lived endoperoxide antimalarials will help circumvent the development of resistance. With improved antimalarial treatments and the eventual development of an effective vaccine, we may one day see a malaria-free world.

Nick Klonis is a Senior Research Fellow andr Leann Tilley is a Professor of Biochemistry and Molecular Biology at the Bio21 Institute, The University of Melbourne.