Nobel Prize

Parasite busters

Print edition : November 27, 2015

Figure 1: The distribution of some of the most devastating parasitic diseases is quite similar and is collectively shown in blue on the world map. Photo: Illustration by Mattias Karlén/Nobel Committee background document, Physiology and Medicine, 2015

William C. Campbell. Photo: Reuters

Satoshi Omura. Photo: Reuters

Youyou Tu. Photo: AFP

Figure 2: Avermectin modified to Ivermectin, which contained two hydrogenation modifications, turned out to be highly effective in both animals and humans against a variety of parasites. Photo: Illustration by Mattias Karlén/Nobel Committee background document, Physiology and Medicine, 2015

Figure 3: (a) A hand-coloured drawing of qinghao in Bu Yi Lei Gong Pao Zhi Bian Lan (Ming Dynasty, 1591 CE). (b) Artemisia annua L. in the field. Photo: Youyou Tu, Nature Medicine, October 2011

Figure 4: A Handbook of Prescriptions for Emergencies by Ge Hong (284–346 C.E.). (a) A Ming dynasty version (1574 C.E.) of the handbook. (b) “A handful of qinghao immersed with two litres of water, wring out the juice and drink it all” is printed in the fifth line from the right. Photo: Youyou Tu, Nature Medicine, October 2011

Figure 5: A model of artemisinin. Carbon atoms are represented by black balls, hydrogen atoms by blue balls and oxygen atoms by red balls. The Chinese characters underneath the model read Qing Hao Su. Photo: Youyou Tu, Nature Medicine, October 2011

The 2015 Nobel Prize for Physiology or Medicine has been shared by William C. Campbell of the U.S., Satoshi Omura of Japan, and Youyou Tu of China for their separate discoveries in treating deadly parasitic diseases like elephantiasis and malaria.

A LARGE number of organisms —viral, bacterial and parasitic —cause deadly diseases in humans and animals. While there was reasonable progress during the 20th century in the prevention and treatment of viral and bacterial infections, developing vaccines and therapies against parasitic infections proved very hard. Compared with the discovery of the antimicrobial action of sulphonamides and other antibiotics in the first half of the last century, there was very limited progress in developing effective therapies for parasitic diseases.

Parasitic diseases have been around for millennia and continue to constitute a major disease burden of the world even today. They affect the world’s poorest populations in sub-Saharan Africa, West and South Asia, Central and South America and even some parts of Europe. Many parasites are also known to cause diseases in domestic animals and livestock. Improving the health and well-being of these populations by developing long-lasting therapies and preventive measures has, for long, been a major challenge for the world medical community.

Parasites, as their very name suggests, reside on, or in, another organism (the host), get their food (nutrients) at the expense of the host, and propagate and cause chronic or life-threatening diseases. There are three main classes of parasites: protozoa (unicellular organisms), helminths (large multicellular organisms, which, in their adult stage, are visible to the naked eye) and ectoparasites (ticks, fleas, lice, mites, etc., that attach to or burrow into the skin and remain there for weeks or months). Malaria, by far the largest killer in the world today despite being the oldest known parasitic disease, is a classic example. All attempts to develop an effective vaccine or a therapy have been unsuccessful. Rapid genetic mutations in the single-celled (protozoan) malarial parasite Plasmodium result in the emergence of drug-resistant variants of the disease within time scales of a couple of decades or even less. Similarly, for many parasitic diseases such as African river blindness (onchocerciasis) and lymphatic filariasis, too, there were no safe and effective therapies for a very long time. These two diseases are caused by roundworms (nematodes), a sub-class of helminthic parasites (Figure 1).

The situation with regard to some parasitic diseases, in particular malaria, changed dramatically in the 1970s, thanks to discoveries made by this year’s Nobel awardees in Physiology or Medicine who discovered “revolutionary therapies” against certain parasitic diseases. One half of the prize went to the 85-year-old Irish-born William C. Campbell, currently a Research Fellow Emeritus at Drew University, Madison, New Jersey, and the 80-year-old Satoshi Omura, Professor Emeritus at Kitasato University, Japan, for the discovery of a new drug, Avermectin, derivatives of which have significantly lowered the incidence of river blindness and lymphatic filariasis. Some of them have also been found to be efficacious against a number of other parasitic diseases. The other half has gone to the 85-year-old Youyou Tu, Chief Professor at the China Academy of Traditional Chinese Medicine, for the discovery of the plant extract Artemisinin. Derivatives of it and combination therapies based on it have been effective in bringing down the mortality rates in recent years among patients suffering from malaria, including its most fatal form, cerebral malaria.

“These two discoveries have provided humankind with powerful new means to combat these debilitating diseases that affect hundreds of millions of people annually. The consequences in terms of improved human health and reduced suffering are immeasurable,” said the October 5 citation by the Nobel Assembly at the Karolinska Institutet in Stockholm, Sweden, which awards the Physiology or Medicine Nobel Prize.

It is estimated that over one billion people worldwide are infected with one or more species of filarial nematodes or worms. Filarial worms, which belong to the family Filarioidea ( phylum Nematoda), usually require two hosts—an arthropod vector (the intermediate host) and a vertebrate (the primary host)—to complete their life cycle. The larval phase occurs within the body of the vector, usually a biting insect. The mature (reproductive) phase occurs in the body of an animal bitten by the insect. There are eight known filarial nematode groups that use humans as their primary hosts. These are divided into three groups depending on the niche region of the body they colonise.

River blindness, or onchocerciasis, which is listed as one of the “neglected tropical diseases” by the World Health Organisation (WHO), is caused by the filarial worm Onchocera volvulus. More than 99 per cent of infected people live in 31 sub-Saharan African countries. The disease also exists in some parts of Latin America and Yemen. The disease is transmitted by repeated bites of infected black flies ( Simulium species). These black flies breed in fast-flowing rivers and streams, mostly in remote villages located near fertile land where people rely on agriculture, and hence the disease name.

Black fly bites introduce O. volvulus larvae in their third stage onto the body of the host, where they penetrate into the bite wound. The infection can cause a variety of conditions, including chronic dermatitis, skin rashes, lesions, intense itching and skin depigmentation. Microfilariae (embryonic larvae), which are released in thousands in the host’s bloodstream by the mature parasite, reach the eye, causing inflammation, corneal scarring and, ultimately, blindness. River blindness is the world’s second leading infectious cause of blindness. An estimated 25 million are infected and more than 300,000 suffer from blindness. The total population at risk from the disease in the endemic countries is estimated to become 250 million by 2016.

Lymphatic filariasis, commonly known as elephantiasis, is an infection of the lymphatic system caused by infection with nematode species Wuchereria bancrofti, Brugia malayi and Brugia timori. Here, the intermediate vector is the mosquito, as against the black fly in the case of river blindness. The microfilariae migrate in a pattern between the deep veins during the day and the peripheral circulation during the night. As the number of nematodes increases over time, the worst effects of this chronic inflammatory disease are felt during adulthood when the damaged and dilated lymphatic vessels cause blockages, swelling of the limbs, fever and immobilisation.

Lymphatic filariasis affects more than 120 million people. It causes chronic swelling and leads to life-long stigmatising and debilitating clinical symptoms, including elephantiasis—oedema with thickening of the skin and underlying tissues —and scrotal hydrocele. This too is classified as a “neglected tropical disease” by the WHO. According to the WHO, currently 1.23 billion people in 58 countries are living in areas where lymphatic filariasis is transmitted and are at risk of being infected. Approximately 80 per cent of these people live in the following 10 countries: Bangladesh, Côte d’Ivoire, the Democratic Republic of Congo, India, Indonesia, Myanmar, Nigeria, Nepal, the Philippines and the United Republic of Tanzania. Globally, an estimated 25 million men suffer with genital disease and over 15 million people are afflicted with lymphoedema.

Avermectin: Combating nematodes

Satoshi Omura, a trained microbiologist with expertise in isolating natural products, worked at the Kitasato Institute in Japan, which had a strong track record in natural product isolation. He came to the United States as a visiting research scientist to the laboratory of Max Tishler, a former director of the Merck Sharp and Dohme Research Laboratories (MDRL), at Wesleyan University, in 1971. Tishler set up a contact between Omura and the MDRL, which had a long-standing interest in antibiotic research. This resulted in a collaboration between H. Boyd Woodruff, the chief microbiologist of MDRL, and Omura, with the aim of discovering new antimicrobial products from microbial fermentation.

Omura’s focus was on a group of bacteria called Streptomyces, which is found in the soil and is known to be the source of myriad antibacterial activities—the antibiotic Streptomycin, for example, was isolated from this by Selman Waksman (Nobel Prize, 1952). However, no new strains of these bacteria had been found. The problem with this group of bacteria was the fact that it was notoriously difficult to culture in the laboratory, causing slow progress in the field. But Omura possessed extraordinary skills in developing unique methods for large-scale culturing and characterising of these bacteria.

Omura succeeded not only in isolating new strains of Streptomyces from soil samples but also in culturing them in the laboratory. From thousands of cultures that he had prepared, Omura selected around 50 of the most promising ones to produce new secondary metabolites with the objective of identifying their antimicrobial activity by further analysis. One of these new strains, which was apparently found near a golf course in Ito, proved to be the strain ( Streptomyces avermitilis) producing Avermectin.

Following the discovery of this new strain, William C. Campbell, an expert in parasite biology and who was then working at the MDRL, acquired Omura’s Streptomyces cultures under an agreement with the Kitasato Institute and explored their efficacy. In the first screening, freeze-dried culture broths were mixed in food and administered to mice infected with the Nematospiroides dubius (a common nematode found in the duodenum and the small intestine of rodents). Though the mice nearly died, one of the cultures showed positive action. By refining the culturing conditions, Omura was able to remove the contaminant, the toxic oligomycin, from the culture broths. Thomas W. Miller, who worked with Campbell, then chemically characterised the active ingredients in the cultures, which included Avermectin B1. Studying the anti-parasitic activity of these agents, Campbell found activity against a variety of parasitic worms in domestic and farm animals. Subsequently, Omura characterised the taxonomic status of the strain through genetic studies and proposed the slightly different name, Streptomyces avermectinius, for it.

In a team effort with other scientists at the MDRL, Campbell later modified Avermectin to a new compound called Ivermectin, which was much more effective than Avermectin against parasitic infections. Ivermectin is a semi-synthetic version of Avermectin B1, with two hydrogenation modifications (Figure 2). Campbell and his associates were later able to show that Ivermectin was (1) extraordinarily potent; (2) active against many intestinal nematodes in various host species; (3) capable of durable long-term activity against some extra-intestinal parasites, including blood-dwelling filarial microfilariae; (4) active against benzimidazole-resistant nematodes (suggesting a different mode of action); and (5) well-tolerated by the host species, suggesting a good margin of safety.

But all the studies until then had been on parasitic infections in animal hosts. Following the positive findings in animal hosts, particularly in reducing dog heartworm, Campbell subsequently proposed that the Avermectin class be investigated in humans with river blindness. In 1981-82, Mohammed Aziz at MDRL, an expert in river blindness, conducted human trials, which were successful. The findings showed that even a single dose of Ivermectin indicated either complete or near-complete elimination of microfilariae, while the adult parasites were untouched.

The drug was also found to be efficacious against W. bancrofti, the parasite causing lymphatic filariasis. In this case too, a single dose sufficed to clear microfilariae from the blood of infected patients. But even now, the mode of action of Ivermectin is not understood in complete detail. It is, however, known that inhibition of ligand-gated chloride ion channels in invertebrate muscle and nerve cells of microfilariae is involved. Though such pathways are present in mammalian nervous systems as well, they seem to have low affinity to the drug and are also protected by the blood-brain barrier in mammalian systems. Ivermectin is thus a very selective drug that kills the parasite at low concentrations without harming humans.

The elimination of river blindness and lymphatic filariasis are key targets for the WHO Regional Strategy to Eliminate Neglected Tropical Diseases. The WHO today regards Ivermectin to be a highly effective treatment for both river blindness and lymphatic filariasis. It has recommended the following regimen for the treatment of river blindness: Ivermectin at least once yearly for about 10 to 15 years. For lymphatic filariasis, a combination therapy with Ivermectin is recommended, comprising a single dose of two medicines given annually to an entire at-risk population as follows: albendazole (400 mg) together with Ivermectin (150-200 microgram/kg) or with diethylcarbamazine citrate (DEC) (6 mg/kg).

The road to the new antimalarial

As mentioned earlier, malaria, a global threat today, is caused by the unicellular parasite Plasmodium. Five species can infect humans, causing shivering, fever and sweating, which occur in repeated episodes as symptoms. The most fatal form is cerebral malaria, a serious condition resulting in encephalopathy (brain disorder), which is generally caused by the species P. falciparum. The other species usually cause milder forms of the disease. According to the World Malaria Report (2014) of the WHO, about 3.4 billion people are at risk of being infected with the disease each year. In 2013 alone, 198 million cases of malaria occurred globally, resulting in 584,000 deaths, the disease burden being the heaviest in Africa.

Until the mid 20th century, malaria was treated with quinine, which was traditionally extracted from the bark of the cinchona tree but later was also synthesised in the laboratory. This was slowly replaced by the newly developed drug, chloroquine. Cloroquine, in combination with the highly efficient insecticide DDT, which could exterminate mosquitoes, came to be adopted as the strategy for eliminating the disease during the 1950s and 60s. However, besides environmental concerns regarding the use of DDT, mosquitoes too began to develop resistance to it. And soon, strains of P. falciparum resistant to chloroquine also began to emerge and spread.

The Nobel Prize-winning drug Artemisinin is a herbal extract from the plant qinghao (the extract is called qinghaosu), which has been described in ancient Chinese literature and has been part of traditional Chinese medicine (TCM) for nearly 2,000 years (Figure 3). The botanical name for the herb is Artemisia annua (sweet wormwood). The Nobel award has, however, prompted several articles in the media, particularly in the U.S., where followers of TCM are on the rise, claiming to various degrees that the award is a vindication of not just the particular drug Artemisinin but the entire system of TCM itself, despite the Nobel committee’s assertion during the press conference that the award does not tantamount to certification of the entire system of TCM itself.

Indeed, on the contrary, as some commentators have pointed out in their blogs, the prize is a vindication of the modern science of pharmacology, specifically pharmacognosy, the branch of pharmacology concerned with finding medicines in natural products. The story of the discovery of the drug Artemisinin is a fascinating one and it needs to be recounted just to drive home the importance of modern scientific approaches in validating traditional medical systems, including Ayurveda.

The resurgence of malaria in tropical regions, including China, because of the emergence of chloroquine-resistant strains of P. falciparum during the 1960s was the time of the beginning of the Cultural Revolution in China. As part of that, the Chinese government, under Mao Zedong, launched a secret military project to develop a remedy against the scourge of malaria. It was not only a major malady affecting China but also Vietnam, which was at war, and the disease was devastating its civilian and military populations. Vietnam too had sought Chinese help to fight malaria.

It was a covert project, named Project 523 to mark the day it was announced (May 23, 1967). Because of the secrecy of the project and the prevailing political climate of the Cold War, none of the scientific papers from the project was published for many years. The earliest ones to be published were in Chinese and not easily accessible to the international community. In fact, many details of the project are still shrouded in mystery, including how Youyou Tu came to be chosen to head the project.

According to a commentary that Youyou Tu wrote in the journal Nature Medicine in 2011 after being awarded the prestigious Lasker Award for medical research, she graduated from the Beijing Medical University School of Pharmacy in 1955, and since then has been involved in research on Chinese herbal medicine. Between 1959 and 1962, she went through a training course in Chinese medicine that was specially designed for professionals with a background in Western medicine. In early 1969, Youyou Tu was appointed head of the Project 523 research group at her institute, which included both phytochemical (study of chemicals derived from plants) and pharmacological researchers.

The group began extracting and isolating constituents from Chinese herbal materials with possible antimalarial activity. According to her, in the first stage of the work, the group investigated more than 2,000 herbal preparations and identified 640 of them with possible activity. By 1971, more than 380 extracts obtained from about 200 Chinese herbs were evaluated against a mouse model of malaria. “However,” she wrote, “progress was not smooth, and no significant results emerged easily. The turning point came when an Artemisia annua L. extract showed a promising degree of inhibition against parasite growth. However, the observation was not reproducible in subsequent experiments and appeared to be contradictory to what was recorded in literature” (emphasis added).

Master stroke

Youyou Tu went back to an intensive review of the literature on TCM. The only reference relevant to qinghao’s ability to alleviate malaria-like symptoms was in Ge Hong’s A Handbook for Prescriptions for Emergencies (340 C.E.). It said: “A handful of qinghao immersed in two litres of water, wring out the juice and drink it all” (Figure 4). This gave her the idea that the standard procedure of boiling and high-temperature extraction could destroy the active ingredient. With this in mind, Youyou Tu redesigned the extraction process using an organic solvent, diethyl ether. “Indeed, we obtained much better activity after switching to a lower-temperature procedure,” she wrote. This brainwave was a master stroke because nothing in the text even remotely suggested cold extraction. Only heating was not specifically prescribed. This new method of extraction also enabled her to separate the extract into its acidic and neutral portions. “At long last,” she wrote, “on October 4, 1971, we obtained a non-toxic, neutral extract that was 100 per cent effective against parasitemia to mice infected with P. berghei and in monkeys infected with P. cynomolgi. This finding represented the breakthrough in the discovery of Artemisinin.” Proponents of traditional medicine systems must recognise and appreciate the scientific reasoning and rational argument that led to the breakthrough, rather than having mere faith in ancient texts and blindly following them without proper validation using a modern scientific approach.

In modern drug development, clinical trials form the cornerstone of determining a new molecule’s safety and efficacy against any disease. In this context, it is important to highlight the fact that even though Youyou Tu was exploring a traditional system, she sought to validate the advocated therapy at every step with modern pharmacological techniques. According to her, during the Cultural Revolution, there were no practical ways to perform clinical trials. So, she and her colleagues bravely volunteered to take the drug themselves to test its safety. After its safety was ascertained, Youyou Tu and her associates went to Hainan province to test its clinical efficacy in 21 malaria patients, about half infected with P. vivax and and the other half with P. falciparum, and these trials gave encouraging results. Youyou Tu found that as against patients administered with chloroquine, those treated with the Artemisinin extract showed rapid decline in fever and the parasite count in the blood.

But, for Youyou Tu, the full characterisation of the drug in modern phytochemical terms was far from over. Her group set out to isolate and purify the active components from Artemisia. In 1972, her group identified a colourless, crystalline substance with a molecular weight of 282 dalton (a unit of mass used in biochemistry; 1 Da = 1.661 × 10 -27 kg), a chemical formula of C 15H 22O 5 and a melting point of 156-157 °C as the active component of the extract and named it qinghaosu (“su” in Chinese means “basic element”).

The next step in Youyou Tu’s journey in her quest for an effective antimalarial was “to move from ‘revelation’ to ‘creation’—that is, to turn this natural molecule into a drug”. As David Gorski, a surgical oncologist, notes in his science blog “Respectful Insolence”: “Pharmacognosy led to the discovery of Artemisinin, but it took medicinal chemistry to turn this compound into a usable, useful drug. Sources rich in the molecule had to be identified, methods of isolating on a large scale had to be developed and a stable formulation had to be produced.” Youyou Tu and her colleagues also succeeded in determining the stereo-structure of Artemisinin with the assistance of scientists from the Institute of Biophysics in 1975 (Figure 5), which showed that it belonged to the class of “sesquiterpene-lactones” (compounds that are sesquiterpenoids, built from three isoprene units, and contain a lactone ring). The structure was first published in 1977 in Chinese.

Describing this final step that is of importance from a therapeutic perspective, Youyou Tu wrote: “We had found that, in the genus Artemisia, only the species A. annua and its fresh leaves in the alabastrum [flower bud] stage contain abundant Artemisinin. My team, however, used an Artemisia local to Beijing that contained relatively small amounts of the compound. For pharmaceutical production, we urgently required an Artemisia rich in Artemisinin. The collaborators in the nationwide Project 523 found an A. annua L. native to the Sichuan province that met this requirement. The first formulation we tested in patients was tablets, which yielded unsatisfactory results.... We shifted to a new preparation—a capsule of pure Artemisinin—that had satisfactory clinical efficacy. The road leading toward the creation of a new antimalarial drug opened again.”

Subsequent clinical trials on 529 malaria cases confirmed that the crystal they had isolated did have the desired antimalarial activity. Many scientists from other institutes too then joined the efforts to improve the extraction procedures and conduct clinical trials. The drug was then given to more than 2,000 patients, some of whom had chloroquine-resistant P. falciparum malaria infection. Further, the drug had cured 131 of 141 individuals with cerebral malaria. Comparative studies on a small number of cases suggested that the drug acted more quickly than chloroquine and there were no reported harmful side effects. However, despite all the success of years of Youyou Tu’s perseverance and painstaking efforts, she also faced challenges regarding the dissemination of the results to the world. “The prevailing environment in China at the time,” notes Youyou Tu, “restrained the publication of papers concerning qinghaosu, with the exception of several published in Chinese. Fortunately, in 1979, the China National Committee of Science and Technology granted us a National Invention Certificate in recognition of the discovery of Artemisinin and its antimalarial efficacy.”

In 1981, Youyou Tu presented her group’s work at the Fourth Meeting of the Scientific Working Group on the Chemotherapy of Malaria, sponsored by the United Nations Development Programme (UNDP), the World Bank and the WHO, in Beijing. Her presentation, titled “Studies on the Chemistry of Qinghaosu”, was a revelation to the world. It drew world attention to the efficacy of Artemisinin and its derivatives in treating several thousand malaria-infected patients around the world. This presentation was also followed with the first English language publication of the work in Journal of Traditional Chinese Medicine in 1982. In 2005, the WHO altered its strategy to combat malaria worldwide to Artemisinin combination therapy (ACT), which is currently used the world over, saving many lives, particularly children in Africa.

Actually, as early as 1973, Youyou Tu had investigated the potential efficacy of its derivative, dihydroartemisinin, which, in her words, “was not initially considered a useful therapeutic agent by organic chemists because of concerns about its chemical stability”. Contrarily, her investigations showed that the derivative was more stable, had 10-fold higher antimalarial activity than Artemisinin and, more importantly, had much less recurrence of the disease during treatment. According to her, adding a hydroxyl group to the molecule also introduced more opportunities for developing new Artemisinin derivatives through chemical modification by esterification. Over the past decade and a half, Youyou Tu’s group has also explored the use of Artemisinin and its derivatives for the treatment of other diseases. The implications of Youyou Tu’s method of researching in TCM, in particular the story of the discovery of Artemisinin, which was grounded in modern pharmacological science, for Ayurvedic prescriptions cannot be overemphasised.

In the context of the uproar over the finding in the West about a decade ago of high concentrations of heavy metals in many samples of Ayurvedic medicines, M.S. Valiathan, the eminent cardiac surgeon and an expert in the Ayurvedic system, observed in an editorial in Current Science: “As long as clinical studies based on the WHO guidelines are ignored and scientific papers remain few, Ayurveda would be handicapped in claiming greater acceptance in India and abroad…. The march from plant extracts and molecules to the market would, however, be faster if the random testing of thousands of compounds against varied diseases were replaced by a selective approach based on clues from traditional knowledge. This is shown by not only the [Indian] examples of rouwolfia and guggul, but also by Artemisinin…. The focus and intensity of this approach would be missing if thousands of compounds from numerous plants are tested for a variety of pharmacological activities. In the former mode, a hunter follows a hot trail whereas the latter represents an angler on a leisurely fishing expedition. Yet, much of the current work for developing drugs from plants follows the angler’s trail…. Ayurveda is applauded but there is little coordination among the stakeholders in regard to clinical research, quality control of herbal medicines or the scientific study of Ayurvedic concepts and practices.... The need of the hour is to put our house in order by coordinating the overlapping efforts… and working for specific targets….”

Unfortunately, there is hardly any evidence to this day of anything of that kind happening. One only hears high-pitched jingoistic noises about our ancient medical knowledge and wisdom. Will the Nobel award signal a change?