Resistance to anti-malaria drugs

Print edition : June 22, 2002

While the number of effective drugs available to control malaria is small, the rapid emergence of drug-resistant strains of the malarial parasites is outpacing the development of new antimalarials.

AMONG the various factors that have contributed to the current resurgence of malaria, growing resistance to antimalarial drugs is perhaps the most important. The rising all-cause mortality rate among African children is attributable directly to malaria, and specifically to the rapidly increasing resistance to antimalarial drugs. Drug resistance is also emerging as a serious problem in the Indian subcontinent. The number of effective drugs available to treat malaria is small and the rate at which resistance is growing is outpacing the development of new antimalarials. Nearly all the antimalarials that are in use today were developed almost 30 years ago and, in general, the pharmaceutical companies, particularly the multinationals, have little interest in developing new cure despite the enormous need.

A Masai tribesman in Africa covers his bed with a mosquito net sprayed with insecticide. The malarial parasite appears to have found new strength, posing a threat not only in Africa but the rest of the world.-SCOTT PETERSON/ GAMMA

There are four species of parasites, all belonging to the genus Plasmodium, that cause human malaria. P.falciparum and P. vivax cause the majority of infections whereas the prevalence of P.ovale and P.malariae is low. P.falciparum is by far the most aggressive species. It has a rapid rate of asexual reproduction and can progress rapidly to severe or complicated malaria, including cerebral malaria, the leading cause of mortality in the malaria-affected population. P.vivax is the most common species throughout Asia, Africa, West Asia, Oceania and South America. In south-east Asia, where renewed efforts at malaria control have shown results, the incidence of P.vivax infection has dropped but that of P.falciparum has remained unchanged.

The main obstacle to malaria control is the emergence of drug-resistant strains of P.falciparum. Resistance is thought to be acquired genetically by the parasite as a result of spontaneous mutations. Mutant clones are selected especially when the parasite is in an environment of sub-therapeutic levels of drug directed against it. According to pharmacology experts, this is especially the case when background immunity is weak and drug pressure high.

Chloroquine is by far the most used antimalarial in conventional malaria therapy. It is an inexpensive and readily available drug in many endemic areas. However, owing to widespread drug resistance, the drug is becoming increasingly ineffective in many parts of the world. In India, the foci of chloroquine resistance have now spread. In a few countries like Thailand, chloroquine resistance is as high as 85 per cent among P.falciparum cases. Chloroquine is now getting increasingly replaced by sulphadoxine-pyrimethamine (S/P) combination, also known by the brand name Fansidar, as the second-line drug. Prior to 1978, S/P drugs resulted in cure rates of 80-90 per cent but since then failure rates have risen to more than 50-60 per cent.

According to the World Health Organisation, resistance to other powerful drugs like mefloquine, halofantrine, pyronaridine and metakelfin have also been emerging and expanding since 1978. In India, reports of chloroquine resistant P.vivax too have appeared in recent years. Indeed, in 1983, in Tanzania, where the drug mefloquine had never been used, a P.falciparum isolate was found to be resistant to the drug. Also, chloroquine resistant strains of P.falciparum in Gabon were found to have reduced sensitivity to mefloquine. It is now believed that resistance to mefloquine could occur even without exposure to the drug because of its synergy with other antimalarials.

Quinine, the alkaloid from the bark of Cinchona tree, which was the first-line treatment for malaria for over 350 years until chloroquine was discovered, is still effective in the treatment of all four forms of malaria. Parenteral formulations of quinine are used in the treatment of severe cases of P.falciparum malaria. In fact, intravenous quinine is considered the only therapy for cerebral malaria. However, owing to high incidence of adverse reactions and its contra-indications in pregnancy and in young children, search for a suitable alternative to treat drug-resistant, complicated and cerebral malaria has been a priority among malaria researchers. Similarly, while Primaquine is effective against latent stages of all malarial parasites and is, therefore, used in the treatment of relapsing malaria, it has the drawback of causing severe anaemia among patients whose red blood cells are deficient in a certain protein. Here too the need for alternative therapy was desired.

Enter qinghaosu or artemisinin, the drug discovered by Chinese scientists in the 1970s. Actually it is a modern antimalarial derivative from an ancient Chinese remedy - the medicinal plant qinghao (Artemisia annua). The earliest description of qinghao herb for treatment of malaria-related symptoms is found in the writings of Ge Hong (281-340 AD). Qinghaosu, meaning the extract of qinghao, was isolated in 1972 from the leaves and flowering tops of qinghao. By the late 1970s several clinical studies in China found qinghaosu to be an exceptional antimalarial agent with negligible toxicity and high efficacy against all forms of the parasite, including drug-resistant ones. Chinese scientists also determined its chemical structure.

The active ingredient of the herb, now known as artemisinin, was found to have this remarkable antimalarial action because of its unique chemical structure, quite unlike previously known antimalarials. In chemical parlance, it is the presence of peroxide (a compound containing two oxygen atoms as -O-O- linear structure known as the peroxy group) forming a kind of bridge in the molecular structure of artemisinin that seems to give the drug its antimalarial activity. (A reduced form of the compound deoxyartemisinin, which does not have the peroxy group, was found to lack the antimalarial activity.)

The artemisinin group of drugs target the parasite when it is in its ring-like form (known as schizont) early in the asexually dividing part in the blood stream stage of its life cycle in the human host. The parasite is thus destroyed before it reaches its immature egg-cell (gametocyte) stage in which form it is ingested by anopheline mosquitoes as blood meal. By their action in the early blood stage, the drugs effectively prevent transmission of drug-resistant forms of the parasite as well. The artemisinin group of drugs thus have 'blood schizontocidal' as well as 'gametocytocidal' action.

The white needle crystals of artemisinin are, however, not soluble in water or oil and, therefore, formulations of artemisinin, other than oral or rectal, are not used. Because of poor solubility, the drug absorption and its bioavailability are also poor leading to its low radical cure rate - in early trials it was as low as 15 per cent. However, since the peroxide bridge is stable under various chemical reactions, several oil- and water-soluble derivatives of artemisinin have since been synthesised, giving rise to a whole new class of antimalarials. These artemisinin derivatives include dihydroartemisinin (DHA), artemether and artesunate, originally developed by Chinese scientists, and arteether and artelinic acid.

Currently, this class of antimalarials is available as the parent compound artemisinin (oral, parenteral and suppository formulations) and as three semi-synthetic derivatives: a water-soluble salt (artesunate) for parenteral or oral administration; and two oil-soluble compounds (artemether and arteether) for intramuscular injection. India has made a significant contribution in this worldwide effort through the development of a highly efficacious variant of arteether, called alpha-beta (a-b) arteether, as an intramuscular injection. Developed by the scientists of the Central Drug Research Institute (CDRI) and the Central Institute for Medicinal and Aromatic Plants (CIMAP), the two Centre of Scientific and Industrial Research laboratories in Lucknow, the drug is being manufactured and marketed by Themis Medicare Pvt. Ltd., Mumbai, under the brand name E-Mal since April 1997.

Artemisinin is cheaper to produce but is intrinsically about five times less active. All other compounds are at least as efficacious as quinine in the treatment of severe and complicated malaria. They lead to faster parasite and fever clearance than other anti-malarials. In spite of the more rapid antiparasitic action of artemisinin compounds, these agents have not been shown to decrease the rate of mortality compared with quinine. These compounds act rapidly against drug-resistant P.falciparum strains but have high relapse rates (about 10 per cent to 50 per cent) when used as monotherapy for less than five days. Artemisinin drugs have a short half-life in the human body and they are eliminated from the body rather quickly (1.6 to 2.6 hours) and so when they are used over short periods (less than five days), clearance of parasites from the blood is only temporary in up to 50 per cent of patients. Recent studies have shown longer durations of therapy (seven days) and combinations of artemisinin derivatives and mefloquine could prevent recrudescence. In Thailand it has been seen that combination therapy (artesunate+mefloquine) resulted in over 90 per cent cure rates of primary as well as relapse P.falciparum infections.

Artemisinin derivatives have been used by over several million patients and are well tolerated. In China these drugs have already replaced quinine as the first-line treatment for P.falciparum malaria. In south-east Asia, combinations of artesunate and mefloquine appear to be the most active drug regimens for treatment of multidrug-resistant P.falciparum. There is no evidence so far of any kind of toxicity in human trials although neurotoxicity has been observed in rats, dogs, and primates when administered repeated doses of artemisinin derivatives. However, owing to their remarkable properties, there are concerns that their uncontrolled and widespread use, particularly as oral formulations, will result in the rapid development of drug resistance to artemisinin as well.

Who has noted that resistance to artemisinin will arise but it is impossible to predict where and when. The increasing use of artemisinin and its derivatives, particularly with unsupervised and incomplete regimens is likely to be a risk factor. Such use is most intense in areas where there are generally low levels of population immunity, and in some areas population migrations leading to temporary exposure to intense transmission. These factors have contributed to the emergence of resistance to other antimalarial drugs and may play the same role in the emergence of artemisinin resistance, WHO has observed.

But given the body of current evidence, since 2001, WHO has begun to recommend the use of artemisinin derivatives as combination therapies (with drugs such as mefloquine) in Africa as first-line therapy where falciparum malaria is widespread. In cases of severe complicated malaria, it has recommended parenteral administration of artemether, the methyl ether of DHA. Several African countries have adopted policies to use artemisinin combination therapy (ACT) for severe drug-resistant malarial infections. The Indian National Anti-Malaria Programme (NAMP) too included artemisinin-based drugs in its drug policy of 1996, which has been retained unmodified in the revised NAMP Drug Policy of 2001.

The NAMP Drug Policy recommends intravenous quinine or parenteral artemisinin derivatives only in severe and complicated P.falciparum cases (for adults and non-pregnant women only) "irrespective of chloroquine resistance status". It has also permitted the use of oral forms of artemisinin derivatives as second-line therapy for treating P.falciparum malaria cases resistant to both chloroquine and S/P combination drugs but made available only against the prescription of a medical practitioner. The CDRI-CIMAP-Themis drug a-b arteether is also part of the NAMP, the Drug Controller-General of India (DCGI) having allowed the drug for use exclusively in hospitals and nursing homes to avoid indiscriminate use.

THE development effort by CDRI-CIMAP scientists began in the 1980s when the plant Artemisia annua was introduced in India by the CIMAP from the Royal Botanical Gardens, Kew, England. Using the agrotechnology developed at the CIMAP the plant was successfully cultivated in the Kashmir Valley to produce large quantities of artemisinin. By genetic upgrading through what is called mass-selection approach, a high artemisinin yielding variety (130 per cent of the yield of the original Kew germplasm) was evolved. Availability of high-quality raw materials in sufficient quantities and at a much cheaper rate laid the basis for the search for a new artemisinin-based drug. Since the plant belonged to undomesticated land races, the CIMAP developed appropriate agrotechnology for the cultivation of A.annua under temperate and semi-tropical climates. The CIMAP has also developed tissue-culture-based micro-clones of the plant, which have shown 3.5 times higher artemisinin yield than seed-raised bulk under field cultivation.

The CIMAP also developed the processing technology for artemisinin from air-dried leaves and flowers of the plant cultivated in Lucknow and Srinagar. Artemisinin is present in low concentration (0.19 per cent) in the plant material. A novel process was developed to extract simultaneously both artemisimic acid and artemisinin. The acid yield in the plant is 8-10 times more than artemisinin itself. A simple and efficient technology was developed to convert the acid into artemisinin with yield up to 40 per cent. This way the overall yield of artemisinin was increased fourfold, thus economising its production cost.

The identification of arteether as the drug to be developed was based on considerations of more potent analogues of artemisinin with better bioavailability. Joint investigations by the CDRI and the CIMAP showed that arteether (ethyl ether of DHA) - as against artemether, which is the methyl ether - was superior to all other derivatives because of its better lipophilicity, pharmacological properties and considerably less toxicity. Arteether comes in two structural forms alpha and beta, known as epimers. The two differ only in the orientation of the molecular structure with respect to the peroxy group. The "magic ratio", as C.M. Gupta, Director of the CDRI put it, "of 30:70 in the epimeric mixture of alpha and beta forms was found to be a potent drug in the pre-clinical antimalarial, pharmacological and toxicological studies carried out at CDRI."

Animal model studies at the CDRI showed strong blood schizontocidal and gametocytocidal activity of a-b arteether as well as no significant toxicity effects. Over seven years (1989-1996), the CDRI successfully carried out Phase I, II and III clinical trials in uncomplicated and complicated P.falciparum malaria cases (based on single 150 mg dose for three days). Phase I trials were carried out at the CDRI (in 20 healthy subjects), Phase II (in 51 patients suffering from P.falciparum infection) at Ispat General Hospital, Rourkela Steel Plant, while Phase III was a multi-centric study at eight centres (Bhilai, Delhi, Dibrugarh, Guwahati, Jabalpur, Jamshedpur, Rourkela and Sonapur) on 257 patients of uncomplicated and 211 patients of complicated P.falciparum malaria in association with the Indian Council of Medical Research's Malaria Research Centre in Delhi.

These trials showed that the drug was well tolerated and there were no adverse effects. The significant aspect of a-b arteether is that a three-dose regimen is sufficient compared to the five-dose regimen required in the standard arteether therapy. Also, the relapse rate was found to be only 5 per cent although a higher relapse rate is seen in other artemisinin derivatives with less than five days' regimen.

The development of the drug cost the CDRI about Rs.2 crores. It was cleared for marketing in 1997 and the process was licensed exclusively to Themis Medicare Pvt. Ltd., Mumbai, for bulk synthesis of a-b arteether. Themis' research and development, with an investment of Rs.50 lakhs, upscaled the lab scale 50g/100g batch size to commercial scale 100 kg batch size production process. The company introduced the drug in the market under the name E-MAL in April 1997. Themis' current production capacity is 30 million doses. The drug is currently marketed at about Rs.300 for the full three-dose regimen, which is considered much cheaper and affordable compared to other drugs for P.falciparum malaria such as artesunate or artemether, which are imported now. Till date about 1.8 million patients have been treated with a-b-arteether in the country. The CDRI has already recovered about Rs.1 crore.

The drug is being used extensively under NAMP since 1999. According to Themis, the value of the total demand for artemisinin derivatives in India is about Rs.32 crores, of which E-MAL has a market share of 35 per cent. Interestingly, NAMP's order of E-MAL during 2000 was only 8.64 per cent of the total demand (Rs.2.7 crores) while during 2001-02 no purchase was made. The NAMP director was not available for comments. On his part, Gupta would like the NAMP drug policy to be amended to prevent use of oral artemisinin derivatives. The policy, he believes, allows indiscriminate use of artemisinin with across-the-counter purchases, which could result in drug-resistance.

A post-marketing surveillance carried out recently by CDRI in association with Themis analysed 300 reports of a-b arteether use from Bihar, Gujarat, Madhya Pradesh, Maharashtra, Rajasthan and Uttar Pradesh. Of these, 257 were P.falciparum cases, 20 vivax, 17 both falciparum and vivax and six were non-parasitic infection. The results showed that 294 cases were cured, five cases improved and one did not show any change in clinical status. Further, the side-effects found in 14 cases were of mild nature.

Among the artemisinin derivatives, at present only artemether (intramuscular injection) and artesunate (oral) as combination therapy are used widely. In fact, in the World Bank-supported effort at procuring artemisinin-based drugs for the Roll Back Malaria (RBM) programme in Africa, WHO has issued a letter of expression of intent to be responded to by prospective drug suppliers by July 2002, which does not include arteether as one of the drugs. This would seem to defy logic because arteether (in India and elsewhere) has undergone trials to the same extent as other artemisinin derivatives. Of the injectables, artemether is the most widely used one because of its stability. But safety of this drug is still debatable, CDRI scientists say. Apparently, a United States study has found that artemether can be biologically broken into methanol, which can undergo oxidation and produce toxic products like formaldehyde and formic acid. The ethyl-form does not have this problem. Interestingly, in 1999 WHO's regional centre in New Delhi purchased the drug worth about Rs.70 lakhs.

According to Gupta, the reason why WHO has not yet approved arteether is because it is developing a pure beta form (b-arteether) under its programme, which has undergone only Phase I trials. The CDRI drug, a-b arteether, is apparently better than the b-form for the following reasons. The CDRI drug has a 30 per cent greater solubility in groundnut oil resulting in a lower - 2ml as compared to 3 ml - injectable dose of the oil; synthesis of a-b form gives 30-40 per cent greater yield; and only a three-day regimen instead of five days for the b-form.

Gupta says: "We are generating some data for WHO's approval of a-b arteether. Basically, these pertain to good manufacturing practices (GMP) as required by the organisation. But WHO is behaving more like a business organisation than a welfare organisation. It would like to see its beta-arteether used in its anti-malaria programmes, and not any other." To explore the export market potential, which both Gupta and Themis believe is large, particularly in Afro-Asian countries, Themis is tying up with Sanofi Winthrop/Sanofi Synthelabo, a French company, which has a large presence in Africa to market the CDRI invention, a-b arteether. "We need not wait for WHO to grant approval," says Gupta.

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