Artemisinin-Based and Other Antimalarials: Detailed Account of Studies by Chinese Scientists Who Discovered and Developed Them

Artemisinin-Based and Other Antimalarials

Detailed Account of Studies by Chinese Scientists Who Discovered and Developed Them

Editors Chinese Edition

Li Guoqiao

Li Ying

Li Zelin

Zeng Meiyi

Editors English Edition

Muoi Arnold

Keith Arnold

Zeng Meiyi

Chinese to English Translation

Yuexin Rachel Lin

Postdoctoral Researcher, Mahidol-Oxford Tropical Medicine Research Unit

Muoi Arnold

Table of Contents

Cover image

Title page

Copyright

The Development of Qinghaosu (Artemisinin)—A View From the Outside

Foreword

Foreword

Preface

List of Contributors

Introduction

Chapter 1. Discovery of Qinghaosu (Artemisinin)—History of Research and Development of Artemisinin-Based Antimalarials

1.1. Discovery of Artemisinin and Early Antimalarial Research

A. The Discovery of Qinghaosu (Artemisinin)

B. Nationwide Collaboration on Artemisinin Research in Treating Malaria

C. Project Validation and the National Invention Award

Acknowledgments

1.2. The Development of Artemisinin and Its Derivatives as Antimalarials

A. Artemisinin

B. Artemether

Acknowledgments

C. Artesunate

D. Dihydroartemisinin

Acknowledgments

1.3. Innovation of Artemisinin-Based Combinations

A. Artemether–Lumefantrine Combination (Coartem)

Acknowledgments

B. Artemisinin–Naphthoquine Phosphate Combination (ARCO)

C. Artemisinins–Piperaquine Combinations (CV8, Artekin, Duo-Cotexin, Artequick)

1.4. China’s Research Program Under the Chinese Steering Committee for Research of Qinghaosu and Its Derivatives

A. WHO Interest in China’s Artemisinin Antimalarials

B. The Chinese Steering Committee for Qinghaosu Research and Its Tasks

C. Joint Projects Between the Chinese Steering Committee for Qinghaosu Research and WHO/TDR

D. Raising the Standards of New-Drug Research and Production Specifications

E. Surveys into Artemisia annua Linnaeus Resources and a Base for Artemisinin Production

F. Preparing for the Global Market

G. Termination of the Chinese Steering Committee for Qinghaosu Research

1.5. Management of Artemisinin Antimalarial Projects

A. Clear Direction and Goals

B. Forward-Looking Innovation

C. New Centralized Management

D. Mutual Aid and Strength in Numbers

E. Respecting Knowledge and Talent

Chapter 2. Agronomics and Biology of Artemisia annua L.

2.1. Propagation and Cultivation of Artemisia annua

A. Botanical and Medicinal Properties

B. Resource Distribution and Origin

C. Propagation

D. Cultivation

2.2. Cellular Engineering of Artemisia annua

A. Overview of Tissue and Organ Culturing

B. Hairy Root Cultures

C. Induction of Shoot Clusters

D. Hairy Roots and Adventitious Shoots Cultured in Bioreactors

E. Establishment of Genetic Transformation System

Acknowledgments

2.3. Biosynthetic Pathways and Bioengineering of Artemisinin

A. Biosynthetic Pathways

B. Genes Involved in Biosynthesis

C. Regulation of Artemisinin Biosynthesis

D. Artemisinin and Precursors Synthesis in Plant Bioreactors

E. Combinatorial Biosynthesis of Artemisinin and Precursors in Microorganisms

F. Other Studies Related to Artemisinin Biosynthesis

Chapter 3. Artemisinin Chemical Research

3.1. Extraction and Isolation

A. The Yunnan Institute of Materia Medica’s #120 Gasoline Extraction Method

B. The Institute of Chinese Materia Medica’s Ether Extraction Method

C. The Shandong Institute of Chinese Medicine’s Acetone Extraction Method

D. The Guangxi Institute of Botany’s and Guilin Aromatics Factory’s #120 Gasoline Extraction Method

3.2. Research Into Other Components of Artemisia annua Linnaeus

A. Terpenoids

B. Flavonoids and Coumarins

3.3. Physical and Spectral Characteristics of Artemisinin

3.4. Determining the Structure of Artemisinin

B. Artemisinin Structure Determined by X-Ray Crystallography

3.5. Chemical Reactions of Artemisinin

A. Reduction of the Peroxide Group

B. Reduction of the Lactone Group

C. Acid Degradation of Artemisinin

D. Alkaline Degradation of Artemisinin

E. Pyrolysis of Artemisinin

3.6. Synthesis of Artemisinin

3.7. Quantitative Analysis of Artemisinins Compounds

A. Methods in the Chinese and International Pharmacopoeias

B. Developments in the Assay of Artemisinins in Body Fluids

Chapter 4. Artemisinin Derivatives and Analogues

4.1. Artemisinin Oil-Soluble Derivatives

4.2. Artemisinin Water-Soluble Derivatives

4.3. Artemisinin Dimeric and Trimeric Derivatives

4.4. Artemisinin Simplified Analogues

4.5. Trioxaquines

4.6. Artemisinins Thermal Stability and Purity Analysis

Chapter 5. Artemisinin and Derivatives: Pharmacodynamics, Toxicology, Pharmacokinetics, Mechanism of Action, Resistance, and Immune Regulation

5.1. Antimalarial Pharmacodynamics

A. Effects on the Malaria Parasite’s Erythrocytic Stage

B. Effects of Artemisinin on Malaria Parasite’s Exoerythrocytic Stage

C. Effect of Artemisinins on Chloroquine-Resistant Strain of Plasmodium berghei In Vivo

5.2. Toxicology

A. Studies in the 1970s

B. Studies in the 1980s

C. Trends in Toxicology Research From the 1990s Onward

D. Conclusions

Acknowledgments

5.3. Pharmacokinetics of Artemisinin and Its Derivatives

A. Artemisinin

B. Dihydroartemisinin

C. Artemether

D. Artesunate

Acknowledgments

5.4. Antimalarial Mechanisms of Artemisinin and Its Derivatives

A. Morphological Effects on Malaria Parasites

B. Effects of Artemisinins on Parasitic Biochemical Metabolism

C. Further Research Developments in Antimalarial Mechanism of Action of Artemisinin and Its Derivatives (Artemisinins)

5.5. Resistance of Plasmodium falciparum to Artemisinins

B. Latest Developments in Resistance Research: Clinical and Genetic

5.6. Immune Regulation Effects of Artemisinin and Its Derivatives

A. Nonspecific Immunity

B. Humoral Immunity

C. Cellular Immunity

Chapter 6. Artemisinin and Derivatives: Clinical Studies

6.1. Artemisinin Clinical Trials

A. Early Clinical Research on Oral and Injectable Formulations

B. Artemisinin Suppositories

6.2. Artesunate

A. Intravenous Artesunate

B. Intramuscular Artesunate in Treating Falciparum Malaria and Comparison With Piperaquine

C. Artesunate Tablets

6.3. Artemether

A. Phase I Clinical Trial

B. Phase II Clinical Trials

C. Phase III Clinical Trial of Artemether-Oil Injections

6.4. Dihydroartemisinin Tablets

A. Phase I Clinical Trial

B. Phase II Clinical Trials

C. Phase III Clinical Trials

D. Conclusion

6.5. Evaluation of Parasite Clearance Speeds and Exploration of Regimens

A. Parasite Clearance Rate and the Concepts of R Fever and T Fever, and R Coma and T Coma

B. Exploration of Artemisinins’ Treatment Regimens

6.6. Artemisinins in Treating Other Parasitic Diseases

A. Schistosomiasis Prevention

B. Piroplasmosis

C. Paragonimiasis

D. Clonorchiasis

E. Toxoplasmosis

Chapter 7. Artemether and Lumefantrine Tablets (Coartem)

7.1. Lumefantrine

7.2. Compound Artemether Tablets (Artemether–Lumefantrine Combination)

Chapter 8. Artemisinin–Naphthoquine Phosphate Combination (ARCO)

8.1. Naphthoquine Phosphate Research

B. Pharmacodynamics

C. General Pharmacology

D. Non-Clinical Pharmacokinetic Studies in Animals

E. Non-Clinical Pharmacokinetics: Action Mechanism

F. Toxicology

G. Naphthoquine Phosphate Tablets Clinical Trials

H. Naphthoquine Phosphate Tablets: Key Clinical Findings, Discussion, and Conclusion

8.2. Artemisinin–Naphthoquine Phosphate Combination

A. Preclinical Pharmacology Studies

B. Clinical Trials

Chapter 9. Artemisinins and Pyronaridine Phosphate Combination

9.1. Chemical Structure and Synthesis of Pyronaridine Phosphate

A. Designing the Chemical Structure of the New Antimalarial

B. Synthesis of Pyronaridine

9.2. Pharmacological Studies of Pyronaridine Phosphate

B. Toxicology of Pyronaridine

9.3. Pyronaridine Clinical Trials

A. Efficacy on Vivax Malaria

B. Efficacy on Falciparum Malaria

C. Efficacy on Cerebral and Other Forms of Severe Malaria

D. Efficacy on Falciparum Malaria in Areas With Endemic Chloroquine Resistance

E. Efficacy on Falciparum Malaria in African Children

F. Efficacy on Plasmodium ovale- and Plasmodium malariae-Infected Patients

9.4. Combination of Artemisinin Derivatives and Pyronaridine in Treating Falciparum Malaria

A. Efficacy of Various Artemisinins and Pyronaridine on Falciparum Malaria

B. Efficacy of Dihydroartemisinin and Pyronaridine on Falciparum Malaria

C. Efficacy of Dihydroartemisinin and Pyronaridine on Chloroquine-Resistant Falciparum Malaria in Africa

Chapter 10. Dihydroartemisinin and Artemisinin in Combination With Piperaquine (Artekin, Artequick); Primaquine and Malaria Transmission; and Malaria Elimination

10.1. Piperaquine and Piperaquine Phosphate

Acknowledgments

B. Antimalarial Activity and Toxicity of Piperaquine

C. Pharmacokinetics of Piperaquine

D. Clinical Studies of Piperaquine

10.2. Dihydroartemisinin–Piperaquine Phosphate Tablets (Artekin)

A. Pharmacology and Toxicology of Dihydroartemisinin–Piperaquine Phosphate Combination (Artekin)

B. Artekin Clinical Trials

10.3. Artemisinin–Piperaquine Tablets (Artequick)

A. Laboratory Toxicology Studies

B. Allergic and Hemolytic Reactions

C. Clinical Trials on Artequick

10.4. Dihydroartemisinin–Piperaquine–Primaquine–Trimethoprim (CV8 Tablets) Clinical Trials

10.5. The Role of Primaquine in ACTs

10.6. Fast Elimination of Malaria by Source Eradication (FEMSE) Using ACTs

Index

Copyright

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The Development of Qinghaosu (Artemisinin)—A View From the Outside

The story of the discovery of qinghaosu is remarkable by any standards. The outside world first learned of this extraordinary Chinese malaria treatment in the late 1970s with the publication in English of a landmark paper in the Chinese Medical Journal. Six small pages reported the discovery, structure, physicochemical characterization, pharmacology, and the laboratory and clinical evidence of antimalarial activity. A plant-derived traditional Chinese medicine seemed about to challenge the products of bark of the cinchona tree (quinine), which has been preeminent in medicine for the past 350  years. The speed of antimalarial action claimed for these new compounds was unprecedented, and they reportedly lacked significant toxicity. Like many malaria researchers when I read this paper I was astounded and eager to learn more and to obtain these compounds for clinical testing. Southeast Asia where I worked was experiencing a rapid decline in the efficacy of the existing antimalarial drugs and a surge in malaria morbidity and mortality. Furthermore, our Chinese colleagues were willing to provide us with the drugs. But the World Health Organization was concerned—the quality of the Chinese products was uncertain—we were strongly advised to wait until WHO could provide us high-quality material for clinical testing. But this never came. Instead WHO TDR embarked on a separate course to develop their own artemisinin derivative—arteether—a compound we learned later had already been synthesized and rejected by the Chinese.

By the late 1980s malaria was causing havoc in rural areas of Southeast Asia, the historical epicenter of antimalarial drug resistance. The available antimalarial drugs (chloroquine, sulfadoxine–pyrimethamine, amodiaquine) had fallen to resistance, and the countries of the region decided they could wait no longer. They decided that they had no option but to import or make the artemisinin derivatives themselves. Trials started and they very soon confirmed all the Chinese claims—artemisinin and its derivatives were indeed the most rapidly acting antimalarial drugs ever. Patients with uncomplicated malaria got better a day earlier, and the responses in severe malaria were equally impressive. Furthermore, the drugs were simple to administer and there was no evident toxicity. Later they were combined with more slowly eliminated antimalarial drugs to form artemisinin combination therapies or ACTs, but despite excellent results from clinical trials with ACTs there was reluctance from international agencies to support these new drugs, and change from the inexpensive and increasingly ineffective treatments they supported. The global malaria death toll continued to rise.

It is sometimes claimed that WHO recommended a policy switch from the failing antimalarial drugs to ACTs in 2001, but this recommendation was very weakly supported, and there was little immediate policy change in Africa, which bears the brunt of the malaria death toll. Eventually the rising tide of antimalarial drug resistance associated suffering and death, and the clear advantages of ACTs over existing drugs which were demonstrated in WHO TDR coordinated multinational trials, forced a change. From 2006, some 35  years after the original discovery of qinghaosu, the new WHO malaria treatment guidelines unequivocally recommended that the world should change to ACTs for the treatment of falciparum malaria. National policies then changed rapidly, and donor support to purchase and deploy the drugs increased. Millions of lives, particularly those of African children, have been saved as a direct consequence.

In the treatment of severe malaria the first large trials outside China were with intramuscular artemether as 30  years ago WHO was interested primarily in a parenteral treatment that could be given easily at a village or health center level. In hindsight this was a mistake. Artemether, which is absorbed slowly and erratically after intramuscular injection, did prove better than quinine—but it was not superior enough to effect a policy change. Eventually very large randomized trials with the water-soluble parenteral artesunate were conducted first in Asia and then in Africa. These showed a large and unequivocal survival benefit with parenteral artesunate, and so artesunate has now replaced quinine as the treatment of choice for severe malaria.

Meanwhile many of the earlier toxicity concerns have been progressively allayed. Neurotoxicity had been shown in beagle dogs after high doses of intramuscular arteether or artemether. This was found to have a pharmacokinetic explanation. There is no evidence for neurotoxicity with current doses of the artemisinin derivatives. From the beginning qinghaosu was shown to affect fetal outcomes in experimental animals adversely. However, careful clinical and experimental studies have shown no evidence of iatrogenic toxicity affecting fetal development or birth outcomes in pregnant women. Today artesunate is the drug of choice for pregnant women with severe malaria at any stage of pregnancy. ACTs are actively recommended for uncomplicated malaria in the second and third trimesters of pregnancy, and the restriction on their use in the first trimester may be lifted cautiously in the near future. Increasingly ACTs are also being used to treat vivax and other human malarias. But there is a darkening cloud on the horizon and that is resistance. It proved very difficult to change susceptibility to qinghaosu and its derivatives in the laboratory, but decades of unregulated use in Southeast Asia did eventually select for resistance, and artemisinin-resistant Plasmodium falciparum parasites are now spreading across the Greater Mekong subregion.

Qinghaosu has been one of the most important antiinfective drugs of the past century, it has saved millions of lives, and it has allowed us again to plan for malaria elimination. It is a discovery worth celebrating. In this comprehensive volume we can now learn for the first time from different perspectives of the details of the early discovery and the extensive and far-reaching research in China that followed.

Professor Sir Nicholas J White, FRS,     Chairman, Oxford University – Wellcome Trust South East Asian Tropical Medicine Research Units

Foreword

Malaria is the most serious parasitic disease worldwide. In the 1960s and 1970s, annual morbidity reached 30–50 million, with around 5 million—mostly children—dying from the disease. The history of mankind’s struggle with malaria is a long one and, in the prevention and treatment of the disease, antimalarial drugs play an extremely important role. The appropriate use of such medications can result in good therapeutic outcomes, as well as eliminate the sources of infection, and prevent its transmission. Beginning with the successful isolation of quinine from cinchona bark in the 1880s, chloroquine was developed in the 1940s, ushering in an era in which chloroquine was the drug of choice for malaria treatment and prophylaxis. In the 1960s, Plasmodium falciparum began to develop resistance toward chloroquine. Morbidity rates soared and mortality was high, especially in Southeast Asia, South America, and tropical and subtropical areas of Africa. This became a severe threat to life and health.

During the Vietnam War, both Americans and North Vietnamese suffered greatly from malaria. Noncombat casualties sometimes outnumbered combat losses. To support North Vietnam, China’s leaders considered this request urgent and called for a task force to research for new antimalarial drugs to aid North Vietnam. The relevant authorities across China formed a Project 523 leading group and set up an administrative body, the Project 523 Head Office, to organize the cooperative efforts of ministerial, military, provincial, metropolitan, and autonomous district research units. They devoted themselves to the research and development of a new antimalarial. At first, compound therapies based on old medicines were devised to meet the pressing need for malaria treatment and prophylaxis. Simultaneously, the researchers directed their energies toward synthesizing new antimalarial compounds and screening remedies in the Chinese materia medica.

In the 1970s, Chinese scientists unearthed and developed artemisinin from the treasure trove of Chinese medical classics. Three artemisinin derivatives with even higher efficacy—artesunate, artemether, and dihydroartemisinin—were subsequently produced. Research into new compounds yielded lumefantrine and naphthoquine phosphate while developing piperaquine further. From the 1990s, these drugs formed the basis of a series of combination therapies designed to raise the cure rate and delay the onset of drug resistance: artemether–lumefantrine, artemisinin–naphthoquine phosphate, and artemisinins–piperaquine, etc. Domestic and international collaboration brought artemisinin-type antimalarials to the world. Today, China’s innovative artemisinin-based combination therapies have become one of the main drugs used worldwide for the prevention and treatment of drug-resistant falciparum malaria. They have been taken up rapidly and broadly to treat tens of millions of cases worldwide, saving the lives of millions of critically ill patients. The discovery of artemisinin and the research outcomes that followed were a significant breakthrough in global antimalarial research and a milestone in the history of the struggle against malaria. They represent an important contribution in this area by Chinese scientists and a model of success for modern medical research in China. In the first 5  years of Project 523, I took part in the screening of antimalarial drugs and in clinical trials in malaria-endemic areas. I experienced the great tribulations of antimalarial research and felt its significance deeply. For what Project 523 has accomplished, I am immeasurably happy.

New drug research is a form of basic science and a medical engineering project that crosses disciplines and specializations. In more than a decade of research under Project 523, scientific, medical, pedagogical, manufacturing, and other units undertook large-scale collaboration. They not only achieved important outcomes in multiple fields but also established a system and management style based on unity in will, goals, direction, and action. With little regard for their personal gain, several hundred scientists and technicians forged ahead cooperatively, maintaining timely and open channels of communication. This spirit, combined with the capable and effective coordinating role of the Project 523 Leading Group and Head Office, was a high point in the world of science and technology. After the conclusion of Project 523, research into artemisinins gradually came to adhere to international standards under the stewardship and coordination of the China Qinghaosu Steering Committee. Research standards improved time and again, and the research and development of new antimalarials attained greater heights.

The new antimalarials developed by Chinese scientists have won multiple high-profile awards and garnered international recognition. Both at home and abroad, researchers have published a large volume of scholarly and scientific works on the artemisinins. The discovery of artemisinin and invention of artemisinin-based antimalarials have received wide-ranging attention internationally. Until now, however, a systematic, comprehensive account from the discoverers and those personally involved in the research is unfortunately still lacking. The publication of Artemisinin-Based Antimalarials fills this gap.

The authors of Artemisinin-Based Antimalarials are all elderly scientists from different units and specialties who took part in the research and development first-hand. The book is divided into 10 chapters, which include a history of the research endeavor and a scholarly scientific record and discussion. In the history section, the events surrounding China’s discovery and research into artemisinins, the scientific standards at different points in time, scientific reasoning and methodology, as well as its administrative, managerial, and collaborative qualities are described extensively and objectively. The scientific achievements reported present the key findings of half a century of antimalarial research in China and a full and accurate summary of scientific data. This is a valuable academic reference work on medical and pharmaceutical research that is of great scientific significance. I hope that its publication will inspire and aid in the research and development of new drugs.

Sun Manji,     Member of the Chinese Academy of Sciences

Foreword

This book is a definitive scientific account of qinghaosu (artemisinin) antimalarial drugs, written and edited by the original scientists involved, and is a major contribution to the academic literature and should serve as a reference source for those interested in antimalarial drug development. It supplements and adds more thorough research background information to the earlier publication, A Detailed Chronological Record of Project 523 and the Discovery and Development of Qinghaosu (Artemisinin), edited by Zhang Jianfang and translated into English (Chinese version published 2006, English version published 2013). This earlier book was written and edited by administrators of the project more as a general history and to correct some false claims being made as to who was involved in qinghaosu’s discovery. Both books give due credit to the Chinese scientists involved in a remarkable achievement, especially since this was achieved under the very adverse conditions of the Cultural Revolution and with much outdated and obsolete equipment.

Artemisinin, its derivatives, and its combinations with other antimalarial drugs are the first-line and most widely used drugs in the world for malaria. This demonstrates how important and amazing this discovery and development has been to the decrease in morbidity and mortality for millions of patients suffering from this disease.

The breadth and depth of the scientific studies described in the following pages clearly demonstrate the competence, hard work, and unreserved commitment to their professional calling of the contributors. These contributors range from basic laboratory specialists in chemistry, physics, botany, pharmacology, and pharmaceutical formulation, to crystallographers, electron microscopists, microbiologists, toxicologists, biologists for animal studies, and clinicians treating patients. But certainly not to be neglected for credit are the contributions to the organization and management of the project by the large numbers of equally competent administrators, such as Zhang Jianfang, Zhou Keding, and colleagues in Project 523. Scientists had to collaborate among themselves and among many geographically widely separated research centers, as well as with the administrators in different locations, who were overseeing the whole undertaking. That this was accomplished in such a short time among such a diverse group of people and under such adverse conditions is to be highly commended.

It has been my privilege and honor to contribute in a minor way to qinghaosu’s development and introduction to the western world by publishing, with Chinese authors, the first clinical research paper in the western medical literature in the Lancet in 1982. And also to be involved in the translation on the artemisinin book written by the administrators, mentioned above and to contribute to this book by the original researchers in the translation and editing of this English edition.

My association with one of the editors and contributors, Li Guoqiao, extends over nearly 40  years, from the occasion when he first introduced me to qinghaosu in 1979 in his laboratory at Guangzhou University of Chinese Medicine. In subsequent years, we have worked together in China and Vietnam to further document the efficacy and safety of qinghaosu, its derivatives and combinations. This collaboration continues today as we work to study and promote his concept of Fast Elimination of Malaria by Source Eradication.

Keith Arnold, MD FACP

Preface

In the 1960s, as the war between the United States and North Vietnam raged ever more fiercely, both sides often needed to reinforce their troop strength. Drug-resistant falciparum malaria was highly endemic in the country. Morbidity and mortality were seriously reducing the number of combat-ready troops. China’s leaders responded to North Vietnam’s call for aid and initiated Project 523, a research program to find a new drug capable of preventing and treating drug-resistant falciparum malaria. They assembled a project leading group and management organization, named Project 523 Head Office. It generated a large-scale collaborative effort involving both military and civilian research units. Based on their experience in the Chinese Materia Medica, scientists discovered the active antimalarial component of the Artemisia annua plant, artemisinin. From this discovery they synthesized and created a series of new antimalarials especially suited to the treatment of drug-resistant falciparum malaria and referred to as artemisinins.

Research into these drugs took place in the 1970s, a troubled time in China. As in a relay event, multiple ministries, disciplines, units, and personnel worked in concert, overcoming one obstacle after another and making progress through extensive collaboration. Of most importance was the finding that the chemical structure of artemisinin was completely different from that of all known antimalarials. The discovery of this compound and the formulation of more artemisinins and the discovery of other drugs were a major milestone in the global history of antimalarial research following the introduction of quinine several hundred years earlier. It also represented an important scientific breakthrough that combined Chinese and Western medicine and advanced the legacy of Chinese medicine itself.

To deal with the relative insolubility of artemisinin, Chinese scientists sought out suitable preparations and, at the same time, changed the structure of the compound. They chemically reduced artemisinin and used its product, dihydroartemisinin, to synthesize artemether and artesunate. These were first developed into four new formulations: artemisinin suppositories, injectable artesunate and artemether, and dihydroartemisinin tablets. Since the 1990s, to delay the onset of drug resistance, improve cure rates, and shorten the course of treatment, artemisinin-type drugs were combined with other new drugs synthesized or invented in China, such as lumefantrine, naphthoquine, and piperaquine. These combinations, termed artemisinin combination therapies (ACTs) by the WHO, have become the first-line worldwide treatment against falciparum malaria. ACTs have been lauded as the hope for the global containment of malaria in the 21st century, and artemisinin-type drugs have already saved the lives of hundreds of thousands, even millions, of critically ill patients.

As a group and as individuals, the scientists involved in this research have won many prizes both in China and abroad; from the State Technological Invention Award, Second Class in 1979—awarded for the New Antimalarial, Artemisinin—other laurels include the State Technological Invention Award, Third Class for artesunate (1989), and artemether (1996); the Outstanding Technological Achievements Group, Artemisinin (1996), from the Qiu Shi Science & Technologies Foundation; and the State Technological Invention Award, First Class for lumefantrine (1990), and Second Class for naphthoquine phosphate (1996). In 2004, the King of Thailand awarded the country’s top prize for medicine to researchers of artemisinin-type drugs. The piperaquine (2005) and artemether (2007) ACTs also won the State Technological Progress Award, Second Class. In 2009 came the European Inventor Award for non-European countries, followed by the Lasker Award in 2011 and the Nobel Prize in 2015.

When Project 523 was first launched, it was a secret program to deal with urgent matters of military preparedness. Most research reports and data were only rarely publicly disclosed, especially those dealing with the history of this research and clinical studies; although they have long attracted the interest of medical historians in China and abroad. Therefore, all this detailed research had never been collected together and made public. Unfortunately as time went on and the people involved aged, this period of arduous struggle was gradually being forgotten or even fabricated and distorted. A detailed and accurate academic work on research on the artemisinins and the other drugs has not been published until now.

In March 2012, a few scientists and leaders who had dedicated their early years to this great work gathered together in the belief that, in their twilight years, it was their responsibility to organize and publish their experiences and their cumulative research data. This would be a legacy to future generations and perhaps provide food for thought for future drug research and development. It would also be the most fitting memorial to Project 523. Forty years ago, when they were in the prime of their lives, these scientists had dedicated themselves wholeheartedly into finding a new drug to prevent and cure drug-resistant falciparum malaria under difficult circumstances. Now in their old age, for neither glory nor gain, they wish to uphold the spirit of cooperation, very prominent at that time, by using their remaining energy to set down a truthful record of those precious years of combined experiences.

Artemisinin-Based Antimalarials is published after three plus years, through joint effort and after several revisions. The editors and writers came from different units and worked in different disciplines. They were either the inventors who had carried out this innovative research first-hand or were key participants and leaders and organizers. The book reflects the relentless creativity of their research and methods, as well as some unique organizational, management, and collaborative techniques. It strives to comprehensively and objectively describe the history of artemisinin-based antimalarial research in China and its technological achievements. The book’s main focus is to introduce China’s research findings, including specialized and detailed data from chemistry, pharmacology, toxicology, and clinical studies. Because of concerns over patents and international collaboration, a portion of this material has not been previously published and is presented here for the first time. Furthermore, the latest international technical and clinical data are also included where appropriate.

Artemisinin antimalarial research has not ceased in China. Beginning in 2003, ACTs were used to study Fast Elimination of Malaria by Source Eradication in Cambodia and Comoros, which yielded promising results. It could provide a new path for the control and eradication of malaria worldwide. Some of the most recent data on this research are assembled in the book.

This book is the first, comprehensive summary of the important achievements made in research into artemisinin-based antimalarials, which has taken place in China for almost half a century. It is a valuable, scientific publication. It can serve as a reference work for new drug research in China, medical education, and for historians of medicine, as well as finding a place in academic libraries and archival collections.

Naturally, as times change and society develops, and as science and technology continue to progress, the research facilities and conditions of the past and our understanding of the unknown are nothing like those of today. Discussion and criticism from different academic viewpoints, and suggestions for improvement on any of the book’s shortcomings, are welcomed.

The Editors

2015

List of Contributors

Editorial Board Members and Contributors

Chen Chang,     National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, China

Guo Xingbo,     Institute of Tropical Medicine, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong Province, China

Jiao Xiuqing,     Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing, China

Li Guoqiao,     Institute of Tropical Medicine, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong Province, China

Li Ying,     Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

Li Zelin†,     Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China

Liang Li,     Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

Liu Tianwei,     CITIC Group Corporation, Beijing, China

Liu Xu,     Guilin Pharma, Guilin City, Guangxi Zhuang Autonomous Region, China

Luo Zeyuan

Yunnan Institute of Materia Medica, Kunming, Yunnan Province, China

Sichuan Institute of Materia Medica, Chengdu, Sichuan Province, China

Ning Dianxi,     Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing, China

Shi Linrong,     National Project 523 Head Office, Academy of Military Medical Sciences, Beijing, China

Wang Jingyan,     Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing, China

Wu Yulin,     Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China

Ye Hechun,     Institute of Botany, Chinese Academy of Sciences, Beijing, China

Ye Zuguang,     Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China

Yuan Shoujun,     Institute of Radiology and Radiation Medicine, Academy of Military Medical Sciences, Beijing, China

Zeng Meiyi,     Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China

Zhou Zhongming,     Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China

Additional Contributors to Chinese Edition

Chen Lin,     Antimalarial Laboratory, Institute of Parasitic Diseases, Second Military Medical University, Shanghai, China

Ding Deben,     Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing, China

Hu Sihua,     Artemisia annua L. Research Center, Institute of Tropical Medicine, Guangzhou University of Chinese Medicine, Guangzhou, China

Li Fulin†,     Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing, China

Li Guofu,     Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing, China

Li Linna,     Institute of Radiology and Radiation Medicine, Academy of Military Medical Sciences, Beijing, China

Li Tao,     Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China

Ma Lina,     Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China

Wang Hong,     University of Chinese Academy of Sciences, Beijing, China

Wang Yiwei,     Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China

Yang Weipeng,     Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China

Zhang Huihui,     Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China

Zhang Xiuping,     State Institute of Pharmaceutical Industry, Shanghai, China

Zhang Zhixiang,     Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing, China

Zhong Jingxing,     Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing, China

Individuals Provided Assistance with Archival and Source Data

Gu Haoming,     Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

Huang Heng

Yunnan Institute of Materia Medica, Kunming, Yunnan Province, China

Sichuan Institute of Materia Medica, Chengdu, Sichuan Province, China

Li Runhong,     Institute for Medical Humanities, Peking University, Beijing, China

Lou Songnian,     Shandong Academy of Chinese Medicine, Jinan City, Shandong Province, China

Tian Ying,     Shandong Academy of Chinese Medicine, Jinan City, Shandong Province, China

Wu Zhaohua,     Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China

Zhan Eryi,     Yunnan Institute of Materia Medica, Kunming, Yunnan Province, China

Zhang Shugai†,     Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

Contributors to English Edition

Richard K. Haynes

Center of Excellence for Pharmaceutical Sciences, North-West University, South Africa

Department of Chemistry, Hong Kong University of Science and Technology, Hong Kong

Tran Tinh Hien

Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam

Nuffield Department of Medicine Oxford University, Oxford, England

† Deceased.

Introduction

Eight years ago, my colleagues and I were seeking out drafts and opinions for our edited volume, A Detailed Chronological Report of Project 523 and the Discovery and Development of Qinghaosu (Artemisinin), from the specialists involved in artemisinin research. Shen Jiaxiang, a member of the Chinese Academy of Sciences familiar with the history of artemisinin, suggested that our book should be accompanied by a scholarly volume on the research into artemisinin-based antimalarials. Now that Artemisinin-Based Antimalarials has been published, jointly written and edited by the scientists involved in the research and development of these drugs, an important milestone has been reached in the history of Project 523. For this, I am sincerely happy.

Project 523 was an urgent assignment handed down by China’s leaders to aid North Vietnam’s war effort. It was a complex and important task, in which time was of the essence. Ministries, commissions, and the military high command adopted the same spirit that had characterized Chairman Mao’s note on the recipe for the atom bomb: active coordination to achieve this task. With the approval of the State Council and the Central Military Commission, the National Leading Group on Research into Malaria Prevention and Treatment (also known as the Project 523 Leading Group) was formed. A head office was also established to organize and coordinate the work. They brought together 50–60 research units across the country, assembling 500–600 scientists and technicians into one formation to carry out research into combating drug-resistant falciparum malaria. Based on China’s traditional materia medica, artemisinin and its derivatives—artemether, artesunate, and dihydroartemisinin—were invented. Using chemical synthesis, a series of new drugs was created and developed, such as pyronaridine, lumefantrine, and naphthoquine. After Project 523 came to an end, the Ministry of Health and the State Administration of Medicine set up the China Qinghaosu Steering Committee to administer and coordinate the further advancement of research into artemisinin-based drugs.

The cooperative efforts undertaken under Project 523 greatly accelerated the Project’s own research work and the development of artemisinin-based antimalarials as a whole. Back then when China’s technical personnel and facilities were relatively lacking, only the mustering of skills and equipment scattered across various departments and units ensured that their research tasks could be achieved in a short span of time. Active collaboration rallied the energies of the entire group, unified plans and programs, allowed for the division of labor, and made up for the deficiencies in any one specialty or facility. If any unit encountered difficulties, many others came to their aid, creating strength in unity and a wholehearted pursuit of their research goals. With the discovery of artemisinin and development of artemisinin-based antimalarials, for example, many different specialties and units were involved, from screening of traditional and regional herbal drugs, discovery of qinghao and its effective extract portion to isolation of qinghaosu (artemisinin), production of a therapeutically effective crystal, and the fashioning of a new drug from the crude traditional medicinal extract. The Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences led the way by obtaining a preparation from Artemisia annua L., which yielded good clinical results, and isolating the effective antimalarial component. This was an important first step, but the clinical trials on the crystals isolated by the Institute did not provide sufficient proof of their antimalarial effects. Research into huanghuahao (the local name for A. annua) by the Yunnan Institute of Materia Medica and Shandong Institute of Chinese Medical Sciences began somewhat later, but they progressed rapidly and an effective crystal was isolated. Shandong lacked the necessary patients to establish the crystal’s efficacy against falciparum malaria. With the Project 523 Head Office playing a coordinating role, Yunnan’s huanghaosu (artemisinin) was transferred to the Guangzhou University of Chinese Medicine’s Project 523 research unit, which was then working on the treatment of cerebral malaria in the Yunnan-Myanmar border region. In 2  months, a basic assessment of the extract could be made. It was highly effective against falciparum malaria and had low toxicity. The discovery of artemisinin as a new antimalarial drug, from crude extract of A. annua (huanghuahao) to the crystal isolated and subsequent clinical evaluation, took only a little more than 2  years. Only 3  years passed before a small amount of artemisinin was manufactured and provided for clinical trials outside China. And only 6  years were needed before batches of the drug were produced on an industrial scale for military use.

The invention of new antimalarial drugs of artemisinin, its many derivatives, and artemisinin-based combination therapies are all the fruits of the Project 523 units’ concerted efforts. Artemisinin itself was appraised at an evaluation conference organized by the National Project 523 Leading Group. The drug was brought before the awards committee of the State Science and Technology Commission by the Ministry of Health and, in September 1979, the Commission issued certifications of invention to six research units: the Institute of Chinese Materia Medica of China Academy of Chinese Medical Sciences, the Shandong Institute of Chinese Medical Sciences, the Yunnan Institute of Materia Medica, the Institute of Biophysics of the Chinese Academy of Sciences, the Shanghai Institute of Organic Chemistry of the Chinese Academy of Sciences, and the Guangzhou University of Chinese Medicine. At the same time, the People’s Daily issued a formal announcement to both domestic and foreign audiences that Chinese scientists had invented a new antimalarial, artemisinin. After artemisinin suppositories, artesunate for injection, and artemether injection had been successfully developed, the Ministry of Health held a press conference on September 11, 1987 to introduce these new preparations. It announced that the suppositories, artemether-oil injections, and artesunate-water injections were especially effective artemisinin derivatives for the treatment of uncomplicated falciparum malaria, and cerebral malaria, and formed the fruits of successful research and development.

In December 2006, in an international conference on research into artemisinins held in Guangzhou, a representative from the WHO said to colleagues from the Project 523 Head Office, Without Project 523, there would have been no artemisinin. Without artemisinin, there would have been no means of saving the lives of millions of people living in malaria-endemic areas worldwide. Thank you!

Artemisinin-Based Antimalarials is a record of the various scientists and technicians who took part in Project 523 and worked tirelessly for more than a decade to aid their brothers and friends in a war of counterinvasion, to enhance the military strength of China’s border defense forces, and to save the lives of people across the globe living with malaria. It is proof of their humanity, their disregard for personal reward, and their bravery in the face of danger. As a member of the Project 523 Head Office, I witnessed the entire development process behind artemisinin and other antimalarials. With various comrades and friends, we toiled hand in glove for 10  years, creating deep friendships. Thinking back on those uncommon times, the invention of artemisinin and other antimalarial drugs has been lauded throughout the world, especially by the people of Asia, Africa, and Latin America, and has brought glory to China. I would like to share this special honor with the authors from Project 523 and their many readers.

Zhang Jianfang,     Former principal administrator, National Project 523 Head Office

Chapter 1

Discovery of Qinghaosu (Artemisinin)—History of Research and Development of Artemisinin-Based Antimalarials

Abstract

This chapter covers in a general way the detailed accounts presented in the succeeding nine chapters.

The introduction describes the malaria morbidity and mortality situation and the request from Vietnam to China for help in treating drug-resistant malaria, which was affecting fighting ability of their troops, a situation also a problem for the Americans during the war 1965–75.

The Chinese therefore, under the personal direction of Mao Zedong, initiated a massive secret research program in 1967 to discover new antimalarials from traditional Chinese medical knowledge, modern, and ancient and from current chemical research efforts.

Artemisinin's extraction from plant material and its studies in animal models, volunteers, and patients, its physicochemical properties, and its structural identification are described.

Artemisinin's derivatives artemether, artesunate, and dihydroartemisinin are discussed as to their properties and in different formulations and in combination with Chinese discovered chemical antimalarials, lumefantrine and naphthoquine and with another chemical piperaquine; an artemisinin-based combination therapy concept to increase efficacy, shorten treatment course, and prevent resistance developing.

An approach to control malaria by attacking the transmitting agent, the gametocyte in humans rather than the mosquito vector, is explained and demonstrated. Extensive coverage is given regarding the organization and efficient management systems, formed by the government (initially Project 523, later the Chinese Qinghaosu Steering Committee) to run the research program, and how it collaborated with the WHO.

Keywords

Development of artemisinins; Discovery; History; Overview; Research

Chapter Outline

1.1 Discovery of Artemisinin and Early Antimalarial Research

A. The Discovery of Qinghaosu (Artemisinin)

B. Nationwide Collaboration on Artemisinin Research in Treating Malaria

C. Project Validation and the National Invention Award

1.2 The Development of Artemisinin and Its Derivatives as Antimalarials

A. Artemisinin

B. Artemether

C. Artesunate

D. Dihydroartemisinin

1.3 Innovation of Artemisinin-Based Combinations

A. Artemether–Lumefantrine Combination (Coartem)

B. Artemisinin–Naphthoquine Phosphate Combination (ARCO)

C. Artemisinins–Piperaquine Combinations (CV8, Artekin, Duo-Cotexin, Artequick)

1.4 China’s Research Program Under the Chinese Steering Committee for Research of Qinghaosu and Its Derivatives

A. WHO Interest in China’s Artemisinin Antimalarials

B. The Chinese Steering Committee for Qinghaosu Research and Its Tasks

C. Joint Projects Between the Chinese Steering Committee for Qinghaosu Research and WHO/TDR

D. Raising the Standards of New-Drug Research and Production Specifications

E. Surveys into Artemisia annua Linnaeus Resources and a Base for Artemisinin Production

F. Preparing for the Global Market

G. Termination of the Chinese Steering Committee for Qinghaosu Research

1.5 Management of Artemisinin Antimalarial Projects

A. Clear Direction and Goals

B. Forward-Looking Innovation

C. New Centralized Management

D. Mutual Aid and Strength in Numbers

E. Respecting Knowledge and Talent

References

Malaria is a mosquito-borne disease caused by the parasite Plasmodium and is widely prevalent in tropical and subtropical areas. The most severe form is falciparum malaria, which can readily lead to severe illness in those with no immunity, such as children, and those living in malaria-free area. Morbidity and mortality are high: In Africa alone, more than a million children per year lost their lives to the disease in the past, but mortality has significantly decreased recently to around 500,000.

Since the 1960s, Plasmodium falciparum has developed resistance to chloroquine and other commonly used antimalarials. This has spread throughout Southeast Asia, South America, and Africa, posing an imminent threat to the control and treatment of malaria worldwide.

Malaria is also an important problem for military medicine. Already in the 1960s, during the Vietnam War, malaria decimated troop strength on both sides. Consequently, the United States of America established a specific Malaria Research Unit under the Walter Reed Army Institute of Research to intensify the study for new drugs. The search for new antimalarials was also conducted in other research institutes around the world including the United Kingdom, France, Australia, and in established pharmaceutical companies in Europe.

To a request from the leader of the North Vietnamese government, Chinese Communist Party Chairman Mao Zedong and Prime Minister Zhou Enlai decided that solving the malaria problem was critical to supporting North Vietnam’s combat capabilities. Thus, China’s State Science and Technology Commission (SSTC) and the General Logistics Department of the People’s Liberation Army (PLA) convened a national collaborative meeting on malaria drug research in Beijing on May 23, 1967. In attendance were officials from the relevant ministries and military organizations and from the provinces (both ordinary and autonomous), cities, and military districts. The top-priority military project that resulted was termed Project 523, after the date of this meeting. The National Project 523 Leading Group was created, with the SSTC at its head and the General Logistics Department of the PLA as deputy. Other members of the Group came from the Ministry of Health; Ministry of Chemical Industries; Commission for Science, Technology and Industry for National Defense; and the Chinese Academy of Science. It was headquartered at the Academy of Military Medical Sciences (AMMS), with corresponding offices at the provincial, city, and military district level. These offices directed the formation, management, and coordination of plans relevant to Project 523.¹

After the development and use of antimalarial combinations for the North Vietnamese troops—malaria prevention and treatment tablets no. 1 (pyrimethamine–dapsone), no. 2 (pyrimethamine–sulfadoxine), and no. 3 (piperaquine–sulfadoxine)—Chinese scientists discovered qinghaosu (artemisinin) from studies on traditional Chinese medicine. Because artemisinin had a novel chemical structure, could effectively and quickly treat falciparum and vivax malaria, and had no cross-resistance to chloroquine, it solved the difficult problem of drug-resistant falciparum malaria. To improve the efficacy and solubility of artemisinin, derivatives such as artesunate, artemether, and dihydroartemisinin were synthesized. Moreover, to delay the emergence of drug resistance, improve cure rates, and shorten treatment time, artemisinin and its derivatives were combined with other antimalarials discovered in China—such as lumefantrine, naphthoquine, and an imitated piperaquine. The combination with pyronaridine, another Chinese creation, was by a Korean pharmaceutical company. The results were a series of artemisinin-based combinations which the World Health Organization (WHO) has termed artemisinin-based combination therapies (ACTs). They have had a significant impact on the global Roll Back Malaria initiative.

This chapter introduces the discovery of artemisinin, the creation of artemisinin derivatives, and the innovation of artemisinin-based antimalarials (ACTs) by Chinese scientists. Under a unified national plan, these researchers worked in tandem and shared resources, with each unit playing important unique roles. In so doing, the National Project 523 Head Office and its successors—the Chinese Qinghaosu (artemisinin) Steering Committee and SSTC—were critical in planning and coordinating the research process.

1.1. Discovery of Artemisinin and Early Antimalarial Research

Zeng Meiyi¹, Li Guoqiao², Li Ying³, Li Zelin¹†

¹Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China; ²Institute of Tropical Medicine, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong Province, China; ³Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

From its beginnings in 1967, the search for a new antimalarial was two-pronged. First, a large number of existing chemical compounds were selected for screening and new chemical compounds were synthesized, resulting in the creation of pyronaridine phosphate, lumefantrine, and naphthoquine phosphate. Second, Chinese traditional medicine was reviewed looking for natural compounds. The 3-year research plan drawn up that year proposed seven traditional medicinal plants as priority subjects for research. Qinghao (sweet wormwood) was among them.²

At that time, some research units had carried out preliminary screening on qinghao but failed to detect antimalarial properties. This may have been due to differences in the species and habitat of the qinghao studied or the extraction methods used. It was subsequently confirmed that only medicinal qinghao derived from the huanghuahao plant (Artemisia annua Linnaeus, of the Asteraceae family) had antimalarial properties. Its active component, qinghaosu (artemisinin), varies significantly depending on whether the plant was obtained from northern or southern China and when it was harvested. The content of qinghaosu is also highest in the leaves of huanghuahao at the growing stage before flower budding. These key factors were only recognized during later research.

A. The Discovery of Qinghaosu (Artemisinin)

1. An Investigation into Traditional Antimalarials: Survey and Screening of Herbs Used in Endemic Areas for Treating Malaria

Chinese herbal medicine may be broadly divided into two categories: remedies described in the historic materia medica or medicine classics, and regional folk herbalism. A specialized collaborative team dealing with Chinese medicine was established by the Project 523 Leading Group. While reviewing traditional Chinese materia medica and folk herbal knowledge, the team also visited areas such as Hainan, the Yunnan border, Guangdong, Guangxi, Sichuan, Jiangsu, and Zhejiang, where malaria is endemic. There, it made extensive inquiries among the local people, gathering their various antimalarial remedies. Samples of medicinal herbs were collected and preliminary preparations made in the laboratory for efficacy and safety tests on animals. These studies were followed by tests on patients at pilot sites to decide which remedies should be explored further.

From 1967 to 1969, for example, an investigative team on folk herbs staffed by members from the Chinese Academy of Medical Sciences’ Institute of Materia Medica (IMM of CAMS), the AMMS, and researchers from Yunnan, Guangdong, and Jiangsu learned of a traditional antimalarial in Guangdong: yingzhao (Artabotrys uncinatus [Lam.] Merr.). After tests and a preliminary clinical trial, it was found to be effective. Under the auspices of Yu Dequan from the IMM of CAMS, the Sun Yat-sen University and its Zhongshan School of Medicine, and the South China Institute of Botany of the Chinese Academy of Sciences (CAS), the active component in A. uncinatus—yingzhaosu A (C15H26O4)—was isolated. Liang Xiaotian, Yu, and others confirmed that this was a new sesquiterpene peroxidic compound.³ Because of the plant’s limited resource supply and the poor stability of yingzhaosu A it was not developed further. Nevertheless, the discovery of yingzhaosu A greatly inspired the subsequent identification of qinghaosu’s chemical structure, which was also of a sesquiterpene peroxide type.

2. Screening of Remedies in Chinese Folk Recipes

In January 1969, the Institute of Chinese Materia Medica (ICMM) of the China Academy of Chinese Medical Sciences (CACMS) in Beijing joined Project 523. The CACMS put Tu Youyou in charge of its 523 Research team, with Yu Yagang as the only team member. Both had degrees in pharmaceutical sciences from Beijing Medical University (now the Peking University School of Pharmaceutical Science) and were trained in modern scientific research methodology. They had also completed a training course at the CACMS, integrating western and Chinese medicine, and understood the historical development of Chinese materia medica.

That April, the department of general affairs of CACMS compiled a mimeographed Collection of Simple and Secrete Antimalarial Remedies with 640 recipes selected from thousands of letters sent by the public over several years.² At the same time, it sent extracts from a few herbal remedies out of the collection to the AMMS for rodent malaria tests. They found that pepper and chilli-alum extracts had an over 80% malaria inhibition rate on rodent malaria. From July to October, Tu Youyou, Yu Yagang, and Lang Linfu carried out on-site clinical trials in Hainan using these two extracts. Out of 44 cases, only one case in each extract group showed parasite clearance. Eventually, these poor results terminated the trial on these extracts.⁴ Then, in early 1970, the National Project 523 Head Office dispatched Gu Guoming from the AMMS, where he was researching herbal antimalarials, to work with Yu to produce herbal extracts and test them on rodent malaria.⁵ Because of her other responsibilities, Tu did not continue with the team but remained at its head.⁶

3. First Indications of Qinghao’s Antimalarial Properties

Yu and Gu continued combing through the Chinese medical literature, preparing and sending herb extracts to AMMS for rodent malaria tests. As his main resources, Yu used the Special Compilation on Malaria—a collection of ancient Chinese antimalarial prescriptions edited by the Shanghai Literature Institute of Traditional Chinese Medicine—and the malaria section of the Qing-dynasty Complete Medical Works of the Library Collection, Ancient and Modern compiled in 1723. He concluded after in-depth study that "black plum, aconite root, shell of fresh-water turtle Carapax Trionycis, qinghao, and others should be singled out, believing that these remedies had been used in isolation and appeared frequently in combined prescriptions and are worthy of multiple animal tests."²

With Project 523 Team as the author, Yu edited a manuscript titled Malaria Remedies in Chinese Medical Literature.² Yu’s manuscript noted that qinghao remedy was first recorded in A Handbook of Prescriptions for Emergencies written by Ge Hong (AD 284–364), prepared thus: "One bunch of qinghao in two sheng of water, mash it and administer the juice. The extracts prepared by him and Gu were tested against rodent malaria by Jiao Xiuqing of the AMMS. Gu reported that Herbs that appeared more frequently among the traditional antimalarial remedies were selected as objects of study. Among them were qinghao, fresh-water turtle shell etc. Extracts were obtained via boiling in water or ethanol extraction, and sent to the screening group for rodent malaria tests. It was found that qinghao (dried aerial part of Artemisia annua L. purchased) had definite antimalarial properties, with an inhibition rate of around 60–90%."⁷

It was Yu and Gu who were the first to experimentally establish qinghao’s antimalarial properties, providing a valuable reference point for their successors. Yu reported qinghao extract’s high–rodent malaria inhibition rate to Tu Youyou.² In September 1970, Tu added qinghao to the screening list.² After that, Yu was assigned other work and left Project 523. Gu also returned to his original unit. From September 1970 to May 1971, ICMM’s antimalarial herbal research was suspended.⁴,⁸

4. Discovery of the Effective Extract of Qinghao Against Malaria

In late May 1971, a Malaria Prevention and Control Research Symposium was held in Guangzhou. The Ministry of Health and National Project 523 Head Office again emphasized the importance of Project 523, prompting the ICMM to redouble its research efforts.⁹

Continuing in her role as team leader, Tu and her team member Zhong Yurong resumed preparation of Chinese medicine extracts.¹⁰ After studying over 100 samples, however, they had to consider selecting new remedies and revisiting those which had already been screened and showed relatively higher antimalarial properties.¹¹ They had previously tested a 95% ethanol qinghao extract, but the rodent malaria inhibition rate was only 40%. The team then turned to ether extraction with better results: Rodent malaria inhibition rates reached 99%–100%, but toxicity was high. After removal of its acidic part, the neutral portion of ether extract obtained had high efficacy and low toxicity.¹² With this, research into qinghao as an antimalarial gained new emphasis.

By March 1972, at a Nanjing meeting of the Project 523 Chinese medicinal herb research group, Tu represented the ICMM in reporting that the neutral portion of qinghao ether extract could achieve a 100% inhibition rate against rodent malaria.¹² This drew the interest of the National Project 523 Head Office and other research units at the meeting.

After toxicology studies in animals and healthy volunteers showed no obvious side effects, Tu and Dai Shaode went to Hainan from August to October 1972 to carry out clinical trials with the neutral portion of qinghao ether extract (coded No. 91). Three treatment dosages were tested and all were effective against malaria, with the high-dose group—total dosage 36  g—yielding the best results. It was effective in treating all 11 cases of vivax malaria, including one case of mixed-species infection. Of nine falciparum malaria cases, seven were effectively treated.¹³ With the Project 523 Head Office playing a coordinating role, the PLA’s Hospital 302 also carried out clinical trials on nine vivax patients; all were cured. The results obtained in all 29 cases were relatively satisfactory.¹⁴

Through animal experiments and clinical trials, ICMM confirmed that the neutral portion of qinghao ether extract had antimalarial properties, especially in treating falciparum malaria. This was a touchstone for further research into qinghao. Armed with this news, the Shandong Institute for Parasitic Disease Control and Prevention tested their own neutral portion of ether extract produced from a local plant, huanghuahao, in October 1972. It was also effective against rodent malaria.¹⁵ In 1973, the Institute used this huanghuahao extract to treat vivax malaria patients on-site, again with good results.¹⁵

5. Discovery of the Active Antimalarial Component, Crystal II, From Qinghao

In August 1972, as Tu was running clinical trials with the neutral portion of qinghao ether extract in Hainan, her colleague Ni Muyun attempted further purification of this neutral portion using aluminum oxide column chromatography but failed to get any significant substance. Zhong Yurong used silica gel column chromatography and gradient elution with ethyl acetate petroleum instead. On November 8, 1972, she isolated three types of crystals numbered I, II, and III.⁴,⁸ Ye Zuguan, of the pharmacology department, carried out rodent malaria tests and found that crystal II was the only one with antimalarial properties. Parasite clearance could be achieved when 50–100  mg of crystal II was fed to mice. Subsequently, ICMM renamed crystal II as qinghaosu II.⁴,⁸

6. Initial Signs of Qinghaosu II’s Efficacy Against Vivax Malaria

The quantity of qinghaosu II produced by ICMM of CACMS via the chromatography method was limited. Hence, right after acute toxicity tests in mice, experiments were run on healthy volunteers. Three of the researchers took qinghaosu II, with one receiving a total dose of 3.5  g over 3  days and the other two receiving 5  g. ECGs, EEGs, and routine liver function, renal function, blood, and urine tests showed no significant changes after medication.¹⁶

Then, from September to October 1973, Li Chuanjie took a team to Changjiang, Hainan, to carry out clinical trials on qinghaosu II. The trial report stated: Three non-local vivax cases were given a total dose of 3–3.5  g in capsules. Average parasite clearance time was 18.5  hours, fever reduction time 30  hours. Follow-up for three weeks, two cases were cured and one was partially effective (parasites recurred at Day 13). Of five non-local falciparum malaria cases, one case was again partially effective (parasite over 70,000/mm³, total tablet dosage 4.5  g, fever reduction after 37  hours, parasite clearance after 65  hours, but parasites reappeared on Day 6). Medication was stopped in two cases due to the appearance of premature heart contractions (one was a first-time infection, parasites over 30,000/mm³, fever subsided at 32  hours after 3  g dosage, but parasites reappeared and temperature rose one day after medication stopped). Two cases were ineffective.¹⁷

This clinical trial provided the first signs that qinghaosu II had some effect on vivax malaria but could not confirm its efficacy against falciparum malaria. From 1974, however, ICMM could no longer isolate qinghaosu II for various reasons and could not run further clinical trials.

7. Huanghaosu’s Efficacy Against Falciparum Malaria in Clinical Trials

Inspired by the results of ICMM of CACMS’ clinical trials with the neutral portion qinghao ether extract, Luo Zeyuan from the Yunnan Institute of Materia Medica (YIMM) tried to find plants from the Artemisia genus with antimalarial properties. From March to April 1973, she collected kuhao (bitter wormwood) and tried extraction with four different solvents: petroleum ether, ether, ethyl acetate, and methanol. Several types of