Current Antimalarial Treatments: Focus on Artemisia annua Dry Leaf

Abstract

Since a lot of drugs that were used for the treatment of malaria has shown resistance to the Plasmodium species. Even the ACT (Artemisia combination therapy) is not effective in certain cases. There is a need to look for some alternatives, which are effective in the clinical treatment of malaria and affordable for the general population. A therapy called Artemisia annua dry leaf antimalarial therapy (ALT) has been shown to be effective against artemisinin-resistant malarial infections and its treatment is resilient to resistance development in animal model systems. This proves to be an effective alternative to presently available antimalarials. This review defines the characteristics of different species of malaria-causing parasites, their vectors, endemicity, and features of the disease development, followed by properties of currently used (approved) antimalarials. The choices and methodologies of administration of antimalarials to adult, child, pregnant, and lactating women patients with acute and complicated malaria are described, followed by strategies to combat drug-resistant malaria, especially artemisinin resistance. A special emphasis on the origin, empirical basis, evidence on clinical efficacy, and cost aspects of ALT is given, along with the focus on the possibilities of repurposing ALT as a treatment for a variety of autoimmune, metabolic, and cancerous diseases.

1. Introduction

Malaria, which results from the transmission of the malarial parasite infection to humans by the bites of infected mosquitoes, is the deadliest infectious disease in tropical and subtropical climates. In recent years (such as 2015 and 2016), 3.5 billion people in 97 countries were at risk of getting infected with the malarial parasite(s). Actually, each of these years, several hundred million humans got malaria-infected and about half a million patients, preponderantly young children, elderly, and pregnant women, succumbed to the disease. In 2016, about 90% of malaria in southeast-cum-south Asia region was contributed by India [1].

In the last about ten years, since the introduction of artemisinin combination therapy (ACT) as the treatment of malaria and regulation of parasite transmission, at least ten countries have become largely malaria-free. During this period, due to success in the control of the disease-causing parasite by chemotherapeutic treatments, such as ACT, prophylaxis, and control of mosquito attacks by use of pyrethroid insecticide impregnated bednets and indoor insect repellents [2], the loss of life from malaria has been halved. For the last 72 years, from the time chloroquine was introduced as a substitute/alternative to quinine in malaria treatment, the disease has been contained by the use of five classes of individual pharmaceuticals (aminoquinolines, aryl-alcohols, including quinolines alcohols, antifolates, hydronaphthoquinone, and endoperoxides) and their combinations. However, malarial parasites have developed genetic resistance against most (perhaps all) of the effective antimalarials and their combinations. Besides, resistant parasites have become geographically widespread. The vector mosquitoes have also developed resistance to insecticides used to impregnate bed nets. The new affordable antimalarial chemical compounds and vaccines undergoing tests and trials are thought to be at least a decade away [3]. All these factors have posed a grave challenge for the control of malarial disease worldwide in the coming years. Discussion is in progress on ways to increase the life span of currently available pharmaceuticals by employing them in alternate combinations, to resist resistance in parasites and to combat parasite transmission. At this time when new effective and affordable malarial treatments are being eagerly awaited, a botanical treatment that appears to clear (artemisinin) resistant malaria has been recently described. Daddy et al., (2017) have reported success in curing 18 cases of severe malaria by administering to the patient tablets made of dry leaves of Artemisia annua (the natural rich source of the pharmaceutical artemisinin) plants. This treatment called Artemisia annua dry leaf therapy (ALT) was found to have cured malaria caused by parasites resistant to currently used antimalarials, including artemisinin derivatives. ALT has been proven to be a safe, efficacious, and affordable antimalarial treatment, with multi-repurposing possibilities [4].

2. Kinds of malaria and symptoms

There are about 200 different unicellular eukaryotic apicomplexans obligate narrow host-range parasite species of the genus Plasmodium, transmitted by dipteran insect species, whose infection can cause various kinds of malarial diseases in a wide range of vertebrates. The five major species of Plasmodium that cause malaria in humans are falciparum (Pf), knowlesi (Pk), malariae (Pm), ovale (Po), and vivax (Pv). The important properties of these human malarial parasites are comparatively summarized (and the references concerned with this section are also given) in Table 1. Among the human malarial parasites, Pk is known to be a zoonotic species whose infection in several species of macaque monkeys produce malaria-like symptoms. Recently another zoonotic species—Plasmodium simium (Ps)—has been found to cause malaria in humans in the Atlantic forest area of Brazil. The natural hosts for Ps are monkeys of the genera Aloulta, Brachyteles, Cebus, and Sapajus [14].

S. No.	Characters		Plasmodium falciparum (Pf)	Plasmodium vivax (Pv)	Plasmodium ovale		Plasmodium malariae (Pm)	Plasmodium knowlesi (Pk)
					curtisi (Poc)	wallikeri (Pow)
1	(A) Features of (n=14) genome	Size (Mb)	23.3	29.1	33.5	33.5	33.6	24.4
2		Estimated gene number	5355	6671	7165	6340	6559	5284
3		G+C content (%)	19	40	29	29	24	39
4	(B) Features of life cycle in human host	Pre-erythrocytic growth in hepatocytes (hepatic schizogony) (number of days = d)	5–7	6–9	8–9		14–16	6–9
5		Whether relapse causing hypnozoites are formed in liver?	No	Yes	Yes		No	No
6		Incubation period (d)	8–15	10–21	12–20		18–60	10–12
7		Fever cycle (erythrocytic schizogony) (number of hours = h)	Tertian (48)	Tertian (48)	Tertian (48)		Quartan (72)	Quotidian (24)
8		Nature of red blood cells affected	All types of erythrocytes	Reticulocytes	Reticulocytes		Mature erythrocytes	All kinds of erythrocytes
9		Size of parasitemia (number of parasites per μL of blood (x10^3)	20–500	20–50	9–10		5–10	0.5–10
10		Whether cytoadherence of parasite cause microvascular dysfunction?	Yes	Rarely (if at all)	Rarely (if at all)		Rarely (if at all)	Yes
11		Whether severe malaria develops?	Yes	Yes	No		No	Yes
12		Whether recrudescence occurs?	Yes (when treatment fails)	Yes (when treatment fails)	Rare		Yes (sometimes after 30 to 50 y from the primary attack)	Yes
13		Time of appearance of gametocytes (d after the start of parasitemia)	8–14	0	0		0	Not known
14	(C) Features of life cycle in mosquito host	Transmission causing Anopheles mosquito vector species	Many species (≥ 70), most prominent are: gambiae, culicifacies and stephensi	Many species (≥ 71), most prominent are: aquasalis, culicifacies, stephensi, darlingi and dirus	Several species (≈ 10), most prominent are funestus, gambiae, stephensi, freeborni, dirus, farauti and atroparvus		Many species (≥ 30), most prominent are: culicifacies, aconitus, arabiensis, atroparvus and freeborni	Several species, including craceus, hackeri, latens and bala-bacensis
15		Time period of sporogony at 28°C (d)	7–12	8–10	12–14		18	12–13
16	(D) Major geographical areas of prevalence		Worldwide tropical and subtropical areas (especially in Africa, Asia, and Mediterranean)	Worldwide subtropical areas (especially in Asia, Latin America, and Africa)	Tropical regions of Africa and Asia and in Pacific islands, sympatrically (subspecies)		Worldwide tropical and subtropical areas (including Pacific islands)	Southeast Asia and South Asia
17	(E) Remarks		Pf is the preponderant cause of malaria. The falciparum malaria is the deadliest and if not treated timely the acute (or un-complicated) malaria turns into cerebral (or complicated) malaria.	Pv can cause severe disease and death due to splenomegaly. The Duffy blood group deficient in Africa when infected are often symptomless	In some cases, relapse can occur as late as 4–5 years from the initial inoculation		It is less life-threatening than vivax and falciparum malaria. However, it can cause chronic lifetime infection	This parasite is zoonotic, also causes malaria in the monkeys Macaca fascicularis, M. nemestrina and Presbytis melalophos. The disease in humans is mild, but can be lethal (mortality≈2%). The Duffy blood group people in West Africa are often insensitive to this parasite. Transmission occurs from humans to monkey and vice versa. Human-to-human transmission is rare (perhaps via the vector A.dirus)

Table 1.

Properties of malaria caused in humans by infection of different species of the alveolate parasite. Plasmodium (Phylum: Apicomplexan; Family: Plasmodiidae) [5, 6, 7, 8, 9, 10, 11, 12, 13].

The insect hosts of Plasmodium species are anopheline mosquitoes. Out of about 515 known species of Anopheles, about 70 are vectors of human malaria [15]. Each of Pf, Pk, Pm, Po, Pv, and Ps are transmitted to humans by several to many Anopheles species, in geographical areas of their occurrence. The genomes of the human malarial Plasmodium species and of the major Anopheles vector species have been sequenced. The vector for Ps has been identified as Anopheles kertezsia cruzii. Phylogenetic distance-wise the parasite species are related to each other as follows: Pf → Po → Pm → Pk, Pv, and Ps [14]. In terms of the frequencies of malaria infections caused by them in humans, the parasites fall in the following order: Pf > Pv > Po, Pm > Pk > Ps. The malaria caused by Pf, Pv, and Pk can be fatal if not treated. The Po and Pm-caused malaria are less severe and generally not lethal. Pv, Ps, and Po-caused infections can remain dormant in the liver for up to many months. Pm infection can remain latent for years. The Duffy blood group deficient (ackr1 = atypical chemokine receptor 1) humans (who are largely the inhabitants of west Africa) are resistant to infection by Pv and Pk because the parasites are unable to invade their Fy a⁻b⁻ erythrocytes [16].

The Pf, Pv, Po, Pm, Pk, and Ps malaria have differential distribution. Pv is the most widespread malaria; it is the major malaria causal parasite in subtropical areas of Asia, America, and Africa. Nearly half of the malaria cases that occur outside of Africa are related to Pv infection. More dangerous than Pv malaria, Pf malaria is predominant in Africa, but also occurs in tropical regions of Asia and in the middle east. Pf malaria is responsible for 90% of the malarial deaths in Africa. The distribution of Pm malaria is similar to that of Pf malaria except that it is much less frequent. Both Po and Pm are the cause of malaria in Pacific islands. There are two subspecies of Po called P. curtisi and P. wallekeri, both are cause of malaria in Africa and Asia, sympatrically. Together, Po and Pm account for about 10 million cases of new malaria each year. Malaria caused by Pk occurs largely in southeast- and south Asia. The Ps malaria is limited to Brazil. In areas where the frequency of occurrence of malaria infections is high, mixed infections of more than one Plasmodium species have been observed [17]. Recently, a rare case of malaria caused by infection of Pf, Pv, Po, and Pm has been reported from a forest area in central India, which has a high incidence of mixed infection [18].

Initial symptoms of malaria are often as nonspecific as one or more of the following types of sickness: fever, chills, sweating, fast heart rate, sore throat, cough, pneumonia, headache, muscular pain, joint pain, fatigue, difficulty in swallowing, hyper-salivation, jaundice, nausea, weakness, vomiting, constipation, and enlargement of the spleen. Laboratory diagnosis is essential to confirm malaria. The most reliable diagnosis is the detection of parasite-infected red blood cells through microscopic examination of thick and thin blood films. The rapid diagnostic tests (RDTs), based on the detection of parasite antigens, can be used, but should not substitute for the needed microscopic tests [19]. Once diagnosed, a confirmed malaria patient should immediately begin receiving the WHO-prescribed treatment at the earliest.

The findings of the microscopic test are helpful in classifying malaria as uncomplicated or severe. In cases of noncomplicated malaria, the parasitemia (% of parasitized red blood cells) is lower than 2%. If parasitemia is 10%, the malarial patient is facing a severe form of the disease. The symptoms of severe malaria include high fever and one or more of the following conditions: renal impairment (dark urine and limited output) acidosis, hypoglycemia, spontaneous bleeding, breathing difficulties, severe anemia, prostration, or coma. Young children and pregnant women are not only more vulnerable to malarial infection but also prone to developing severe malaria. Consequences of severe malaria in a pregnant woman include miscarriage, stillbirth, premature birth, and birth defects in neonates. Generally, all kinds of malaria cause bone loss due to chronic bone inflammation and adversely affect the functioning of skeletal and heart muscles due to poor supply of nutrients and oxygen [20]. There occurs macrovascular dysfunction in Pf and Pk malaria due to adherence of infected cells to walls of blood vessels [21]. The above kinds of deficits imposed by malaria, span of morbidity, possibility of death can all be checked by antimalarial drug treatment, which also aims to clear malarial parasites from the body of a malarial patient such that malaria does not relapse and transmission to mosquitoes is blocked. Antimalarial drugs are also used as chemoprophylaxis, in mass drug administration campaigns to limit the spread of malaria in endemic areas, and for travelers visiting the malaria-endemic areas.

3. Currently used antimalarial drugs

The antimalarial drugs are: quinine, mefloquine, halofantrine, and lumefantrine (aryl aminoalcohols); chloroquine, amodiaquine, and piperaquine (4-aminoquinolones); primaquine (8-aminoquinoline); pyronaridine (mannich base); atovaquone (naphthoquinone); proguanil, pyrimethamine and sulfadoxine (antifolates); tetracycline, doxycycline, and clindamycin (antibiotics); and artesunate, dihydro-artemisinin and artemether (artemisinin-derived endoperoxides). Table 2 gives the chemical structures, purpose and regimen of administration to malaria patients, biological effects on Plasmodium parasites, and prescription properties. There is large difference in interclass and intraclass properties of the drugs. In vivo half-life of artemisinins is short (0.5 to few hours) as compared to that of lumefantrine, pyronaridine, pyrimethamine, sulfadoxine, piperaquine, and chloroquine (3 to 60 days). Quinine and artemether are highly insoluble in water and are usable for parenteral application. Artemisinins are very fast-acting drugs. Quinine, chloroquine, piperaquine, and artemisinins are able to block the transmission of parasites to mosquitoes. Primaquine too blocks transmission but also prevents Pv and Po malaria relapses. Quinine, mefloquine, lumefantrine, atovaquone, and artemisinins do not allow the multiplication of parasites in mosquitoes. Unlike proguanil, pyrimethamine, sulfadoxine, and atovaquone target singular but different parasite functions, whereas artemisinin derivatives, chloroquine, and quinine exemplify antimalarials, which target multiple functions in parasites.

Table 2.

Structure, activity, and related features of different classes of antimalarials, that are presently in use against various developmental stages of Plasmodium falciparum (Pf), Plasmodium vivax (Pv), Plasmodium ovale (Po), Plasmodium malariae (Pm), and Plasmodium knowlesi (Pk) caused human malaria(s) and genetic markers of resistance detected against the antimalarials in P.falciparum and P.vivax.

a–i = Antimalarial resistant drug alleles: a, mdr1 = Multi-drug resistance gene on chromosome 5; b, crt = Chloroquine resistance transporter gene on chromosome 7; c, nhe1 = Sodium/hydrogen exchanger gene on chromosome 13; d, mrp1 = Multi-drug resistance-associated proteins gene on chromosome 1; e, plasmepsin-2, -3 = Aspartyl (protease plasmepsin gene located on chromosome 4; f, cyt b = cytochrome B gene on mitochondrial genome; g, dhfr = Dihydrofolate reductase gene on chromosome 8; h, dhps = Dihydropteroate synthetase gene on chromosome 4; i, kelch-13 = Kelch 13 propeller gene located on chromosome 13, (the gene product has three domains: an apicomplex domain, a BIB/POZ domain, and a β-propeller Kelch domain).

To overcome deficiencies of individual chemotherapeutics and to slow down resistance development, antimalarials are now used in combinations. The following combinations have been recommended by WHO to cure various kinds of malaria (Table 2): chloroquine + primaquine (against Po and Pv malaria); quinine + tetracycline or clindamycin (against severe malaria); ACTs = artemether + lumefantrine or mefloquine, dihydroartemisinin + piperaquine, artesunate + pyronaridine or sulfadoxine + pyrimethamine or artesunate + amodiaquine (against uncomplicated malaria, especially those caused by Pf). Primaquine or alternatively tafenoquine is given additionally to stop relapses and transmission; both are contraindicated for G6PD deficient patients. Tertian malaria caused by Ps is curable by chloroquine + primaquine treatment [14]. The combinations used for chemoprophylaxis in endemic areas are atovaquone + proguanil and proguanil + chloromycetin. For chemoprophylaxis mefloquine and doxycycline are also used preferably singly.

4. Treatments based on currently used antimalarial drugs

Table 3 presents a summarized account of the first-line treatments for acute and severe malaria caused by different species of malaria parasites in adults, pregnant women, and young children, as recommended by WHO. The recommendations are a result of scores of trials carried out, for the cure of a different kind of malaria in endemic areas of their occurrence in Africa, America, Pacific islands, southeast and south Asia, on adult men and women, pregnant and lactating women and young children. Some of the references on which the WHO recommendations are based are given at the bottom of Table 3. The dosages of drugs for children are to be adjusted to body weight. Some drugs are prescribed when malarial patients suffer from concurrent ailments or inherited metabolic deficiencies, the proscriptions for each of the antimalarial drugs are given in Table 2. Importantly, primaquine is not to be administered to pregnant and/or breastfeeding women. Because severe malaria patients can suffer from a blockage in blood flow, filling up of fluid in lung’s air sacks, clotting in blood vessel, renal failure, and/or seizures, etc., they must be treated in intensive care environment. Any concurrent bacterial infection in malaria patients should receive immediate attention, along with malaria treatment.

S. No.	Indicative malarial condition	Malaria caused by
		Plasmodium falciparum	Plasmodium vivax, P.ovale, or of these mixed with P.falciparum	P.vivax, P.ovale, P.malariae or P.knowlesi
1	(A) Uncomplicated malaria: (i) in adults	Oral: Artemether + Lumefantrine (the drug(s) of choice)^a ; Dihydroartemisinin + Piperaquine (not to be administered to patients suffering from cardiac condition(s))^b; Atovaquone + Proguanil (known to produce pronounced gastrointestinal side effects)^c; Quinine + Doxycycline^d; and a single dose of 0.25 mg/kg body weight of Primaquine on the first day or 15 mg/Kg of methylene blue for 3 days	Oral: Artemether + Lumefantrine^a; Dihydroartemisinin + Piperaquine^b or Chloroquine^m	Oral: Chloroquine^m
2	(ii) in pregnant women	Oral: Quinine + Clindamycin (in: all trimesters)^e; or Artemether + Lumefantrine (in all trimesters)	As in column 2; item 2; or Chloroquine^m	As in column 3; item 2
3	(iii) in children (< 12 years)	Oral: Quinine + Clindamycin^e; Atovaquone + Proguanil^f; Artemether + Lumefantrine^g; or Dihydroartemisinin + Piperaquine^h	Chloroquine^n	Chloroquine^n
4	(B) Severe or complicated malaria: (i) in adults	Intravenous Artesunate for 24h or more (or until the patient can swallow tablets, but not more than 5 days)^i, followed by a full course of Artemether + Lumefantrine^a or of Dihydroartemisinin + Piperaquine^b or Intravenous Artesunate treatment^i followed by a full course of Quinine + Doxycycline^d; or alternatively Intravenous Quinine^j for 48 hours or until the patient is able to swallow tablets, followed by oral Quinine + Doxycycline^k	As in column 2; item 4; or intravenous artesunate treatment followed by the full course of Chloroquine	As in column 2, item 4
5	(ii) in pregnant women	Intravenous Artesunate^i, followed by Artemether + Lumefantrine or Dihydroartemisinin + Piperaquine as in item 4 above; or Intravenous Quinine followed by a course of Quinine + Clindamycin^l	Oral: Chloroquine (in all trimesters); Artemether + Lumefantrine (in all trimesters)	As in column 2, item 4
6	(iii) in children (≤ 12 years)	A rectal suppository dose of upto 100 mg (10 mg/kg body weight) Artesunate followed by intravenous Artesunate or Quinine and thereafter dihydroartemisinin + Piperaquine or Quinine + Clindamycin; as for adults with dosage adjusted as per body weight	As in column 2	As in column 2
7	Relapse (prevention): (i) in adults	Not applicable	Primaquine^o (not to be administered to Glucose-6-phosphate dehydrogenase = G6PD deficient) ^p	As in column 3
8	(ii) in pregnant and breastfeeding women	As above	Chloroquine^q followed by Primaquine upon withdrawal of breastfeeding	As in column 3

Table 3.

The prevalent antimalarial treatment regimens against un-complicated and complicated malaria(s) in adults, pregnant and breastfeeding women, and children [5, 28, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67].

a = 4 tablets (such as of Coartem) followed by 4 tablets at 0, 8, 24, 36, 48, and 60 hours; b = 3 or 4 tablets (such as of Eurartesim) daily for 3 days; c = 4 tablets (such as of Malarone) daily for 3 days; d = 600 mg Quinine sulfate every 8 h for 5–7 days and 200 mg doxycycline daily; e = 600 mg quinine sulfate every 8 h plus 450 mg clindamycin every 8 hours for 7 days; f = 1 to 4 Malarone pediatric tablets (as per body weight from ≤ 10 kg to ≥ 40 kg); g = 1–4 tablets at 0, 8, 24, 36, 48, and 60 hours (as per body weight from ≤ 15 kg to ≥ 35 kg); h = ½ to 3 tablets, followed by equal amount at 24 and 48 hours (as per body weight ≤ 10 kg to ≥ 60 kg); i = 2.4 mg/kg body weight injection of artesunate at 0, 12, and 24 h and thereafter daily; j = starting dose of 20 mg/kg body weight of quinine hydrochloride in 5% dextrose over a 4 h period, followed by 10 mg/kg body weight of Quinine hydrochloride every 8 h for upto 48 h and later every 12 h; k = 600 mg quinine sulfate three times a day for 5 to 7 days from the start of quinine therapy, plus oral 200 mg of doxycycline each day for 7 days; l = intravenous quinine therapy to be followed by oral quinine, like, except in place of doxycycline, clindamycin (450 mg) will be administered three times a day for a period of 7 days; m = 620 mg at 0 h, 310 mg at 8 h and 310 mg on day 2 and 3; n = 10 mg starting dose, then 5 mg/kg at 8 h and also on day 2 and 3; o = 15 to 30 mg/day or 0.2–0.5 mg/kg body weight/day for 14 days depending on body weight; p = The G6PD deficiency may be administered by 0.75 mg/kg of primaquine per week for 8 weeks; q = 500 mg each week.

The options for the treatment of uncomplicated malaria in adult men and women are artemether + lumefantrine; dihydro-artemisinin + piperaquine; atovaquone +proguanil; quinine + doxycycline. Along with a drug combination, a dose of primaquine ensures control of the transmission. The treatment for Pv, Po or mixed malaria is one of the following: artemether + lumefantrine; dihydro-artemisinin + piperaquine; chloroquine. For Pm and Pk malaria, the drug recommended is chloroquine. The drug options for children against Pf malaria are: artemether + lumefantrine; dihydroartemisinin + piperaquine; atovaquone + proguanil; quinine + clindamycin. Chloroquine is the drug recommended for children against Pv, Po, Pm, and Pk malaria. Pregnant women afflicted with any kind of malaria are recommended to use quinine + clindamycin or artemether + lumefantrine, and those having non- Pf malaria are also recommended chloroquine. The treatment options for patients with severe malaria in adult men and women caused by all kinds of parasites are intravenous artesunate for one or more days until the patient can swallow tablets, but not more than 5 days, followed by a full course of artemether + lumefantrine, dihydro-artemisinin + piperaquine or quinine + tetracycline; or intravenous quinine for 2 days or until the patient can begin to swallow tablets, followed by a full course of quinine + doxycycline. In the severe malaria cases caused by Pv or Pk, the intravenous treatments are to be followed by a full course of chloroquine. Pregnant women patients with severe Pf malaria are to be given intravenous artesunate or quinine treatments, like that for adult men and women. Whereas artesunate-treated pregnant women patients are to be given a full oral course of artemether + lumefantrine or dihydro artemisinin + piperaquine, those who received intravenous quinine will be given a full oral course of quinine + clindamycin. The severely ill pregnant women, with any non-Pf malaria, will be given a full course of oral chloroquine or artemether plus lumefantrine, irrespective of the trimester of pregnancy. Young children with complicated malaria are to be first treated with artesunate given rectally followed by the treatments (with dose adjustment according to the patient’s body weight) recommended for severely ill adult patients.

5. Strategies proposed to treat and control multidrug-resistant malaria

The studies summarized in Table 2 shows that resistance has developed against the antimalarial drug in current use singly or in two-drug combinations. It is visualized that in the absence of new drugs and vaccines in the near future, there is an urgent need to use the existing drugs in better ways and in new combinations. The two treatments advised for chloroquine-resistant Pv malaria are: (a) dihydroartemisinin + piperaquine with a dose of primaquine [66], and (b) administration of verapamil, the calcium channel blocker which serves as a chemo sensitizer, along with chloroquine to improve drug efficiency. The possible treatments advised for ACT-resistant Pf malaria are (a) A new ACT combination of artesunate + pyronaridine to be introduced as a treatment. (b) ACTs, such as dihydroartemisinin + piperaquine and artesunate + mefloquine be used rotationally (c) The period of use of prevalent ACTs be extended from 3 days to up to 7 days. (d) ACTs be used as combinations of artemisinin drugs with two partner drugs, such as artemether + lumefantrine + amodiaquine, and dihydroartemisinin + piperaquine + mefloquine. (e) The double and triple drug ACTs be used sequentially. (f) The combination of fosmidomicin and piperaquine serves as a sure cure. Another important suggestion is the administration of a dose of the drug ivermectin in the endemic areas along with the ACT or singly periodically on a mass scale. Ivermectin taken by mosquitoes along with the blood meal of ivermectin administered to humans will have a killing effect on them, thereby drastically controlling malaria transmission [67, 68, 69].

An entirely new strategy to treat multi-drug (ACT) resistant malaria has been developed wherein tablets made of dried leaves of the A. annua plant (natural resource of artemisinin drugs) are used [4]. The origin and essential features of this highly affordable malaria therapy are discussed below.

6. Artemisia annua dry leaf antimalarial therapy (ALT)

The ALT has been earlier called the whole Plant based artemisinin combination therapy (pACT). pACT was called a combination therapy because of the involvement of artemisinin and other metabolites present in the leaves of A. annua in the antimalarial therapeutic effect of Artemisia annua dry leaves [70, 71]. ALT is unlike the conventional ACTs (mentioned in Tables 2 and 3), in which the artemisinin component, extracted from A. annua or artemisinin synthesizing transgenic tobacco or Physcomitrella patens whole plant [72], or semi synthesized from Artemisia annua produced natural precursor(s) [73] is present in its derived pharmaceutical forms, such as artesunate, artemether, and dihydroartemisinin. ALT is a non-pharmaceutical antimalarial treatment that depends on artemisinin and many other metabolites naturally biosynthesized and present in the leaves of the Artemisia annua plant, but for many of which the mode(s) of antimalarial action remains to be revealed. To get WHO recommendation, ALT has to go through extensive and essential fundamental and clinical research which needs to demonstrate that ALT is safe, efficacious, and would not promote the development of resistance to artemisinin in malarial parasites.

ALT uses standardized tablets (Figure 1) as the antimalarial drug prepared by compressing the dried pulverized leaves, harvested from cultivated plants of a specific variety(ies) of Artemisia annua, which contain ≥1% artemisinin.

Figure 1.

ALT tablets made from dry Artemisia annua cv Sanjeevani leaves. a = A. annua freshly harvested leaf; and b = Tablets made by compressing the dried A. annua leaves.

The origin of ALT, as a dependable medicine against multi-drug-resistant malaria, is based on information from historical texts and a number of experimental findings. Some of the important empirical basis for ALT is annotated below:

1. There is recorded evidence that the Chinese people have been using A. annua material as a remedy for fever and chills, such as those associated with malaria. One of the effective materials consumed in traditional medicine was the consumption of the juicy extract of water-soaked A. annua leafy stems. The Chinese traditional medicine literature does not report any case of resistance development against A. annua treatment used [74].

2. The A. annua plant material has been used by human populations in various parts of the world where the species existed naturally for various purposes, including for medicinal uses and as an item of food for livestock and humans without notice of any harmful effects [75] and therefore the species has been granted the GRAS (Generally Recognized As Safe) rating. Accordingly, A. annua leaves in amounts ≤ 30 g dry weight/day can be safely consumed [76].

3. In a study batch of healthy mice were orally fed on the one hand with an amount of artemisinin in its pure form, and on the other hand, were fed an equal amount of artemisinin in the form of A. annua dried leaves. The blood stream of mice fed with dry leaves contained > 40 times more artemisinin as compared to mice fed with pure artemisinin. Mice were required to be fed with > 45-fold more pure artemisinin (as a component of the normal mouse food) than artemisinin in dry A. annua leaves, so that artemisinin could be detected in the mouse bloodstream [77].

4. In another study, it was observed that oral administration of the Artemisia annua leaves to the Plasmodium chabaudi—infected mice killed the parasite without causing toxicity to mice. It was further found that parasitemia in the infected mice was reduced at least five-fold more by a single dose of A. annua leaves as compared to an equivalent dose of pure artemisinin, and the effect of dry leaves lasted longer than that of pure artemisinin (Elfawal et al., 2012). The experiments at c and d above suggested that the presence of metabolites other than artemisinin in the dry leaves of A. annua improved both the bioavailability of artemisinin in the bloodstream and the therapeutic efficacy of artemisinin in infected red blood cells. These possibilities were evidenced by correlating the phytochemistry of A. annua leaves with the response of healthy and parasite-infected mice to the feeding of pure artemisinin versus A. annua leaves, as above and below in e and f. Recently, using CaCo-2 model of intestinal transport, the digestates of A. annua dried leaves were found to improve the artemisinin transport by 37% [78].

5. The leaves of A. annua plants are known to contain a number of classes of secondary metabolites including artemisinic compounds other than artemisinin. Many of these possess varying levels of anti-plasmodial activity, albeit much weaker than in artemisinin. The non-artemisinin, antimalarial compounds affect the survival of parasites via mechanisms that are independent of that for artemisinin or which determine the availability or activity of artemisinin at its site(s) of action. Some of the metabolites of Artemisia annua characterized for possession of their own kind of anti-Plasmodium activity, according to their chemical class, are as follows [79, 80]: artemisinic compounds = arteannuin B, artemisinic acid, dihydroartemisinic acid; coumarin = scopoletin; flavonoids = artematin, casticin, circilineol, chrysoplenetin, chrysophenol-D, eupatorin, kaempferol, luteolin, myrcetin, quercetin; phenolic acids = chlorogenic and rosmarinic acids; saponins; sulfated polysaccharides; terpenes = artemisia alcohol, artemisia ketone, borneol, camphene, camphor, caryophyllene, 1, 8-cineole, germacrene D, limonene, myrcene, nerolidol, α-pinene, phytol, sabinene, spathulenol, α-terpineol. The flavonoids and phenolic acids in general inhibit the cytochrome enzymes, present in the liver and intestine, that metabolize artemisinin to deoxyartemisinin, thereby increasing the bioavailability of artemisinin in the bloodstream [81].

6. ALT was shown to be effective against artemisinin-resistant malarial infections and its treatment was resilient to resistance development in animal model systems. Administration of a single oral dose of A. annua dry leaves (24 mg artemisinin/kg body weight) to rodents infected with artemisinin-resistant P. yoelli cured their parasitemia, whereas an equivalent dose of pure artemisinin proved to be ineffective on corresponding animals. It was further shown that the stable resistance to A. annua dry leaf treatment, in P. chabaudi infected mice, occurred 2.7 times slower than the acquirement of resistance to pure artemisinin. Achievement of resistance to dry A. annua leaf treatment in P. chabaudi-infected mice was found to be 1.6 times lower than that for the treatment with artesunate + mefloquine (ACT) [82].

7. The clinical use of ALT treatment on human patients with severe Pf malaria in the Democratic Republic of Congo proved the efficacy of ALT. For ALT treatment, tablets of 500 mg weight, each containing 5.5 mg artemisinin, were prepared by compressing powdered dry leaves of Anamed-A3 variety of A. annua. The patients given the ALT treatment were 6 males and 12 females, from 14 months to 60 years of age, whose malaria did not cure from treatment with artemether + lumefantrine, nor from intravenous artesunate treatment. The malaria patients had entered the severe phase which included symptoms, such as loss of consciousness, convulsions, frustration, shock, respiratory distress, pulmonary edema, bleeding, gastric distress, and jaundice. Among the patients, the adults were administered one tablet twice daily for 5 days, children of 5–15 kg body weight and 15–30 kg body weight, were given quarter and half tablet twice daily for 5 days, respectively, and those in a coma or too young to swallow tablets, the tablet-dose was crushed, mixed with water and delivered via nasogastric tube. All the patients got cured of their malarial disease and there were no adverse side effects. In ALT treatment on another set of patients, rectal administration of dried pulverized leaves of Artemisia annua was found effective in curing Pf malaria [83, 84]. More extensive studies are needed that will cover 28 days of follow-up after treatment with ALT.

From the evidence described above, about the roles of diverse phytochemicals present in the leaves of A. annua in augmenting the inhibitory/lethal effects of artemisinin in ALT on infections of Plasmodium species on animal model systems and about clinical efficacy and safety of ALT on human malaria patients, it is possible to conclude that ALT is an inexpensive but safe and effective option for treating acute and severe malaria. Since multiple secondary metabolites with the independent lethal mode of action on malarial parasites are involved in the efficacy of ALT, it is possible to further conclude that it will take a considerable time period before any resistance evolves against ALT treatment in malarial parasites or via it against artemisinin. It has been advised that the safety of ART treatment in pregnant women be evaluated and that nausea resulting from oral intake of dry leaf tablets may be controlled by encapsulation or use of anthelmintics or sweet substances [85]. Should there be recrudescence, the ALT treatment may be repeated or alternatively a triple ACT treatment be given.

7. ALT: establishment of the compositional consistency of tablets

Like for pharmaceuticals, stringent control over the quality of A. annua dry leaf tablets, during their manufacturing process, is essential for ALT’s inclusion in the first line of antimalarial therapeutics. To achieve this objective in practice all the individual steps of the process must be standardized. To obtain leaves of high artemisinin content, only the identified genotypes of A. annua be grown under consistent and specified cultivation conditions. To retain the secondary metabolites in high concentrations, the harvested shoots of field-grown plants must be dried under clean and ambient conditions [86] to retain the secondary metabolites present in them in high concentrations. From the dry shoots, leaves are to be mechanically separated from the stem on clean surface, the dry leaves produced from different fields should be homogenized, sieved, pulverized using a blade cutter or equivalent instrument, characterized, and converted into tablets of standard weight, size, and content of artemisinin and a few flavonoids and terpenes, under hygienic conditions [80].

The A. annua crops can be cultivated in temperate and subtropical agroenvironments, such as those available in the countries of central and southern Europe, central Asia, southeast Asia, south Asia, east Africa, South America, and in Australia. Several genetically improved and bred varieties of wide adaptability, whose leaves upon drying contain 0.7 to 1.2 % artemisinin, are readily available, including Anamed (A3), Artemis, CPQPA, Jeevanraksha, Arogya, and Sanjeevani [87]. Besides, several to many seed industry-bred varieties of A. annua are also available.

In India, Jeevanraksha was developed as a product of a polycross hybrid of Asha variety x a Chinese accession followed by back crossing with Chinese parent, selected for ≥ 0.5% artemisinin content in vegetative stage leaves in subsequent generations. Arogya was a selection of globular-shaped hyper-branched segregants from Jeevanraksha lines that had adapted to the temperate agroclimate of Kashmir having ≥ 0.8% artemisinin content in vegetative stage leaves. Sanjeevani was developed as a polycross product of Arogya x Jeevanraksha selected for ≥ 1.0% artemisinin content in leaves of the vegetative stage (Sushil Kumar, personal communication).

A. annua is a short day-flowering, open-pollinated annual shrubby species that completes its life cycle in upto one year time. The sowing and harvesting times of A. annua crops to obtain high-quality produce of leaves has been prescribed according to the agro-climates of country-wise geographical locations of cultivation and variety(ies) [87]. The nursery-grown plants of one month or more of age are transplanted in fields @ 20–70 thousand plants/ ha, depending on the plant architecture and average field duration of plant population of the variety used. Nursery plants are raised by spreading the seeds on a wet soil surface, in farmyard manure fertilized field. The number of seeds required for planting 1 ha of the crop is 3–5 g. Fields of sandy-to-sandy loam soil type are used and fertilized with manure and fertilizers @ N:P: K: 60:40:40 kg/ha. The transplanted A. annua crop, to produce dry leaves for ALT, is harvested before flowering occurs on plants. The plant shoots are dried at temperatures ≤ 40 °C, in the field, under shade, or in specially designed temperature-controlled chambers. The desirable moisture content in the dried leaves is 10–12%. Dry leaves are stored and transported in the form of large blocks by compressing the leaves in molds.

Artemisia annua has been in commercial cultivation by farmers in India for more than 15 years, under the public-institution (CSIR-CIMAP) assisted farmer-company (IPCA) partnerships. In recent years such farmer-company partnerships have covered 2500 h/y, largely in north-west India and in this region preponderantly in the Indo-Gangetic plains area. A.annua is also being cultivated in central and southern India. As a result, India has become a major resource of artemisinin and its derivatives. According to the agroclimate of the Indo-Gangetic plains, the most suitable time for the sowing of the nursery is 15 December to 15 January. Seedlings are transplanted in to the fields vacated by potato crops between 20 February and 1 March. This summer crop of A. annua is harvested between May 28 and June 5 (several weeks before the onset of monsoon rains) and shoots are dried under shaded conditions. Alternatively, or additionally, the plants growing in the nursery are transplanted in fields vacated by wheat crop from 15 May onwards, and the resulting crop is harvested between 21 September and 1 October (after the withdrawal of monsoon rains and with the onset of inflorescence development, but before flowering occurs). The autumn crop is dried in temperature-controlled chambers. The yield of dry leaves from the summer and autumn, harvested crop is 2.5 and 3.5 T/ha, with 0.8 to 1.2% artemisinin content, respectively, depending on the variety used; the highest levels of ART (1–1.4%) are present in the leaves harvested from the crops of Sanjeevani variety (Sanjay Kumar, Ramesh Srivastava, and Anil Gupta, personal communication).

Need is felt internationally for new genotypes of A. annua and for methodologies of plant population propagation such that the individual plants under cultivation have the same genotype or largely similar genotypes. Since A. annua is an open-pollinated crop, individual plants in populations of its registered varieties Anamed (A3), Jeevanraksha, Sanjeevani, and others demonstrate phenotypic differences arising from the segregation of alleles of thousands of genes which are present in heterozygous condition. A genomic study has confirmed the presence of heterozygosity at a large number of protein-coding genes, among 63226 genes identified in A. annua. The quality of dry leaf tablets from any available variety is the result of an average phenotype of its cultivated populations. In the future it is desirable to have ALT tablets from plants of a single genotype. There are several possibilities to pursue this aim. One of these is to develop elite inbred lines through selfing in existing varieties for 6 or more generations. The seeds of the chosen inbred line will be always produced in isolation. Second, F1 hybrids of two selected inbred lines, selected for heterosis, may be chosen for cultivation. Again, F1 seeds will be produced from co-cultivation in isolation of the parental inbred lines whose own seeds will be produced in isolation. Special genotypes, an important one being photo-period independent early flowering, could be developed in the background of chosen singular genotype(s). When suitable genotype(s) have become available for mono-genotype-culture, an alternative method to produce planting material on a mass scale could be the deployment of micro-propagation procedures [88, 89]. Any one selected plant from Jeevanraksha, Sanjeevani, or Anamed (A3) could become a clonal variety with the use of micropropagation for genotype multiplication.

8. Cost-effectiveness of ALT treatment

The ALT treatment in comparison to ACT treatment is highly cost-effective. In the Indo-Gangetic plains area, the cost of cultivation, harvesting, and processing of harvested shoots to obtain dry leaves of A. annua var Jeevanraksha, Arogya or Sanjeevani (all genetically related), and profit for farmers, under the farmer-private company partnership scheme, for two hectares of crop yield of 50 tons of dry leaves is ∼ Rs. 2, 00, 000 (or ∼US$ 3,500). The cost of producing 10 million tablets of 500 mg dry leaves each can therefore be speculated as ∼ Rs 5,00,000 (or ∼ US$ 8500). Considering the expenditure of all kinds on the supply chain of ALT tablets, the cost of a 10 tablets treatment for an adult is estimated as less than Rs 1 (or less than US Cents 17). The ALT treatment in India will be at least 60 to 150-fold less costly than an ACT treatment. It is possible to conclude that large-scale adoption of ALT treatment as advised above can tremendously advance the aim of WHO and 97 malaria-endemic countries, including India, to significantly reduce or eliminate the burden of malaria by 2030. ALT capsules have the added advantage of being used as suppositories.

9. Possibilities of using ALT beyond malaria

A variety of disease conditions in humans and livestock are known to respond curatively to artemisinic-, terpenoid-, and flavonoid-compounds present in A. annua leaves. There is thus a strong possibility that ALT tablets may prove to be of therapeutic value against many diseases beyond malaria. There is robust evidence that demonstrates that many viruses-, bacteria-, fungi-, protozoa-, and helminths- caused infectious diseases on the one hand and autoimmune-, and digestive systems/ metabolic- disorders, and cancers on the other hand are attenuated/ prevented by treatment with artemisinins and A. annua leaves [90].

The drugs artemisinin and artesunate have been found to inhibit replication/multiplication of hepatitis causing hepatitis B (HBV) and C (HCV) viruses and sore-inducing herpes virus and it is close relative cytomegalovirus in cultured human cells [91]. The in vitro growth of Mycobacterium tuberculosis (the bacterium which causes tuberculosis in humans), as well as the tubercular bacterial growth in infected mice, has been found to be arrested by artesunate. The addition of artemisinin to the culture of Aspergillus fumigatus (which causes aspergillosis in human) has been observed to stop the growth of fungus. Artemether and extracts of A. annua leaves have proved lethal to in vitro growing Acanthamoeba castellani (a cause of amoebiasis in humans) [92]. Treatment of mice infected with Acanthamoeba with water-, alcohol- or chloroform- extract of Artemisia annua leaves was observed to have increased the life span of diseased animals. Feeding of A. annua leaves to the broiler chickens infected with Eimeria tenella parasites saved the infected animals from the development of coccidiosis disease [93]. Growth of both visceral and cutaneous leishmaniasis causing Leishmania parasites, in human macrophage cultures, was found to be attenuated by the treatment of artemisinin. Analogously, the leishmania infections in model animals were also observed to have been arrested by treatment with artemisinin or A. annua leaf powder. Artesunate was observed to inhibit the Toxiplasma gondii infection of cultured human cells and of mice in vivo. Trypanosomiasis (human African sleeping sickness) like disease caused by Trypanosoma brucci infection in experimental mice and rats were found to have been cured by artemether treatment. Artemisinin and artesunate treatments given individually inhibited the growth of T. brucci and T. cruzi (the cause of chagas disease in humans) in cultured human cells. Infection in humans and in experimental mice of Schistosoma mansoni, as well as S. japonicum (both the species are cause of schistosomiasis disease), was observed to get inhibited by treatment with each of the drugs- artemether, dihydroartemisinin, and artesunate [94].

In different studies, artesunate was found to cure/suppress and relieve symptoms of collagen-induced rheumatoid arthritis, Crohn’s disease, ovalbumin-induced asthma, and lipopolysaccharide-induced uveitis, all in model animals. Obesity and fatty liver diseases caused by consumption of a high fat/ nutrition diet in experimental animals were found to be cured by treatment with A. annua leaf extracts. The A. annua leaf extract also cured alloxan-induced diabetes in rats. It was found that artemether treatment, to type1 diabetic zebrafish, mice and rats, and human pancreatic islets, transformed the pancreatic α cells into β cells such that insulin synthesis started relieving the type 1 diabetes symptoms [95]. Cells of human cell lines of pancreatic-, hepatocellular-, gastric-, colorectal- and renal- cancer stopped proliferating and got killed by an oncosis-like process upon treatment with artesunate. Also, the xenographs of pancreatic-, hepatocellular-, gastric- and renal-cancers in animal models were found to regress upon treatment with artesunate. The artemisinin treatment produced analogous results in in vitro and in vivo gall bladder cancer and in in vitro cervical cancer. The experimental findings that artemisannua controlled obesity and diabetes in model animals strongly suggest that ALT as a treatment for these diseases in humans [87].

Clearly, the above discussion suggests that the mechanisms of biological actions of artemisinins and artemisannua are such that these agents serve as broad-spectrum therapeutics, such as to cure a variety of human diseases. These observations raise the possibility that perhaps ALT can substitute for artemisinins and artemisannua and ALT can be a therapy for multiple diseases beyond malaria. In view of the above, the need for pilot studies and clinical trials on quality-controlled ALT tablets for studying the response of their administration to patients of each of the different nonmalarial, as well as malarial diseases, that respond to artemisinins and artemisannua, cannot be overemphasized.

10. Concluding remarks

In the last ten years, the incidence of malaria disease was reduced by 20% and mortality among malaria patients by 30%. This was in the main achieved by the use of two-drug ACTs and chloroquine in the treatment of falciparum and vivax malaria, respectively, and by the use of primaquine treatment to block the transmission of parasites from humans to mosquitoes (Table 3). However, the falciparum and vivax malarial parasites have developed genetic resistance against a large majority of the approved antimalarial pharmaceuticals in some of their populations in malaria-endemic areas, thereby making the drugs ineffective (Table 2). There has been independent development of artemisinin resistance in southeast Asia and Africa; consequently, ACT treatments too have become ineffective in parts of these geographical areas. To meet the challenge of multi-drug resistant falciparum malarial strains, treatment with three-drug ACTs has been advised. This year a new treatment (ALT) has been added to cure the acute and complicated malaria caused by ACT-resistant falciparum parasites. The ALT treatment comprises capsules filled with or tablets made from A. annua dry leaf powder, derived from cultivated plants of specific variety(ies) bred for ≥ 1% artemisinin content and a combination of other therapeutically active metabolites naturally present. A regimen of two 500 mg leaf powder tablets a day for 5 days was found to cure adults suffering from ACT-resistant complicated falciparum malaria that was unresponsive to ACT or iv artesunate (most likely artemisinin-resistant). The ALT treatment’s malaria curing property has been related to antimalarial activities of artemisinin, several other artemisinic compounds, many terpenes and flavonoids, and other types of molecules present in the dry A. annua leaves. ALT is safe and seems resilient against artemisinin drug resistance development. The cost of an ALT treatment was estimated to be about 100-fold lower than that of an ACT treatment. Extensive putative use of ALT has gained importance since a recent policy statement of WHO emphasizes the importance of affordability for everyone of safe, efficacious, and quality medical products. The ALT, besides being an efficacious antimalarial treatment has properties that raise possibilities of its multi-repurposement as a treatment against all those diseases which respond curatively to artemisinin, its derivatives and A. annua leaf powder or its extracts. This list includes diseases as diverse as hepatitis, tuberculosis, leishmaniasis, toxoplasmosis, trypanosomiasis, schistosomiasis, asthma, rheumatoid arthritis, diabetes, and cancers of various body organs. There is now an urgent need for (a) further evaluation of artemisinin efficacy against several of the listed diseases in vivo models, and (b) pilot studies and clinical trials to attest ALT treatment for varied malaria and diseases beyond malaria for which artemisinin efficacy has been experimentally established, for the benefit of billions of patients of above-listed diseases.

Acknowledgments

The author is highly grateful to the Director KIET Group of Institutions, Dr. (Col) A. Garg; Joint Director KIET Group of Institutions, Dr. Manoj Goel and Principal KIET School of Pharmacy, Dr. K. Nagarajan; Emeritus Scientist, Prof. Sushil Kumar for their continuous inspiration, guidance, and support.