Section 5 Malaria Treatment

#### **Chapter 9**

## Current Antimalarial Treatments: Focus on *Artemisia annua* Dry Leaf

*Richa Goel*

#### **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 drugresistant 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.

**Keywords:** malaria, *Artemisia annua* dry leaf antimalarial therapy, currently used antimalarial drugs, drug-resistant malaria, *Artemisia*, artemisinin

#### **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 southeastcum-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].

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


#### *Current Antimalarial Treatments: Focus on* Artemisia annua *Dry Leaf DOI: http://dx.doi.org/10.5772/intechopen.106736*



**Table 1.** *Properties of malaria caused in humans by infection of different species of the alveolate parasite.* Plasmodium*(Phylum: Apicomplexan; Family: Plasmodiidae) [5–13].*

#### *Current Antimalarial Treatments: Focus on* Artemisia annua *Dry Leaf DOI: http://dx.doi.org/10.5772/intechopen.106736*

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 ab* 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, hypersalivation, 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.

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


#### *Malaria - Recent Advances and New Perspectives*

**146**


#### *Current Antimalarial Treatments: Focus on* Artemisia annua *Dry Leaf DOI: http://dx.doi.org/10.5772/intechopen.106736*



#### *Current Antimalarial Treatments: Focus on* Artemisia annua *Dry Leaf DOI: http://dx.doi.org/10.5772/intechopen.106736*


*Malaria - Recent Advances and New Perspectives*


 **2.** *Structure, activity, and related features of different classes of* 

Plasmodium

*detected against the* 

 vivax *(*Pv*),*

Plasmodium

*antimalarials*

 *in*

P.falciparum *and*

 ovale *(*Po*),*

*antimalarials,*

Plasmodium

P.vivax.

 malariae *(*Pm*), and*

 *that are presently in use against various* 

Plasmodium

 knowlesi *(*Pk*) caused human malaria(s)*

*developmental*

 *stages of*

Plasmodium

 *and genetic markers of resistance*

 falciparum *(*Pf*),*

#### *Current Antimalarial Treatments: Focus on* Artemisia annua *Dry Leaf DOI: http://dx.doi.org/10.5772/intechopen.106736*



*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.*

#### **Table 3.**

*The prevalent antimalarial treatment regimens against un-complicated and complicated malaria(s) in adults, pregnant and breastfeeding women, and children [5, 28, 58–67].*

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.

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 [67–69].

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.

*Current Antimalarial Treatments: Focus on* Artemisia annua *Dry Leaf DOI: http://dx.doi.org/10.5772/intechopen.106736*

#### **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.

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:

a. 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

#### **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.*

Chinese traditional medicine literature does not report any case of resistance development against *A. annua* treatment used [74].


*Current Antimalarial Treatments: Focus on* Artemisia annua *Dry Leaf DOI: http://dx.doi.org/10.5772/intechopen.106736*

> 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].


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-tosandy 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 temperaturecontrolled 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

#### *Current Antimalarial Treatments: Focus on* Artemisia annua *Dry Leaf DOI: http://dx.doi.org/10.5772/intechopen.106736*

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 soreinducing 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.*

#### *Current Antimalarial Treatments: Focus on* Artemisia annua *Dry Leaf DOI: http://dx.doi.org/10.5772/intechopen.106736*

*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 malariaendemic 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 ACTresistant 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.

### **Acknowledgements**

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.

### **Author details**

Richa Goel Department of Pharmacognosy, KIET School of Pharmacy, KIET Group of Institutions, Ghaziabad, UP, India

\*Address all correspondence to: richagoel06@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Current Antimalarial Treatments: Focus on* Artemisia annua *Dry Leaf DOI: http://dx.doi.org/10.5772/intechopen.106736*

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[93] Dragan L, Gyorke A, Ferreira JF, Pop IA, Dunca I, et al. Effects of *Artemisia annua* and *Foeniculum vulgare* on chickens highly infected with *Eimeria tenella* (Phylum Apicomplexa). Acta Veterinaria Scandinavica. 2014;**56**:22

[94] Akande F, Fagbemi B. *In vivo* and *in vitro* effects of artemisinin group of drugs on trypanosomosis in mice. Journal of Natural Sciences Engineering and Technology. 2011;**10**:73-80

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#### **Chapter 10**

## Combating Antimalarial Drug Resistance: Recent Advances and Future Perspectives

*Samuel Uzondu, Adaeze Echezona, Chinekwu Nwagwu, Adaeze Onugwu, Lydia Ugorji, Chinazom Agbo, Franklin Kenechukwu, John Ogbonna, Paul Akpa, Petra Nnamani, Audu Momoh and Anthony Attama*

#### **Abstract**

This chapter X-rayed antimalarial drug resistance (ADR) by plasmodium species with a particular focus on *P. falciparum*, which is the most deadly species of the malaria parasite responsible for over 90% of the global malaria burden domiciled in Sub-Saharan Africa. The introduction intently looked at malaria therapeutics across the decades and the development of drug resistance by the parasite. With the malaria parasite (*P. falciparum*) as the focal point, the mechanisms by which they develop resistance to antimalarial drugs was looked at, including factors affecting drug resistance development. Armed with this knowledge, the chapter also highlighted the therapeutic interventions taken against this hydra-headed monster together with their limitations and recent advances towards addressing those limitations or opening new frontiers for research exploration. Future perspectives that will provide research strategy and direction as possible tools for combating drug resistance development by the malaria parasite were also discussed.

**Keywords:** antimalarial drug resistance (ADR), *P. Falciparum*. Malaria, mutation

#### **1. Introduction**

Malaria is a public health concern which has ravaged majorly Sub-Saharan Africa which accounts for over 90% of the global malaria burden [1]. This disease is caused by Plasmodium species, five of them are known to cause the disease in man. They *are Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium knowlesi. Plasmodium falciparum* is the responsible for the most deadly form of the disease found in Sub-Saharan Africa causing the highest global

morbidity and mortality rates in Africa. Children from 0 to 5 years and pregnant women are the most vulnerable to this disease. There has been a lot of therapeutic interventions by way of drugs targeted at eliminating or eradicating malaria. Prior to the introduction of the first synthetic drug chloroquine, cinchona alkaloids, quinine and quinidine have been used for the treatment of malaria. Chloroquine was introduced in 1940 as the mainstay for malaria treatment for about a decade plus until the parasite developed resistance to it. This led to the development and introduction of other drug classes consecutively as follows.


The following classes of drugs consecutively have been used clinically in the treatment of malaria; (1)4-aminoquinolines (e.g. chloroquine, amodiaquine and piperaquine) (2) aminoalcohols (e.g. mefloquine, halofantrine and lumefantrine) (3) antifolates (e.g. Sulphadoxine, pyrimethamine, proguanil) (4) hydroxynaphthoquinone (e.g. atovaquone) and (5) endoperoxides [e.g. artemisinin and derivatives with their combination with other antimalarial drugs (ACTs.)] Classes 1–4 are no longer used as first lines as a result of resistance development to them by the malaria parasite. Currently the endoperoxides' clinical efficacy against the malaria parasite is being threatened by emergence of artemisinin-resistant and ACT-resistant strains of the parasite particularly in the Greater Mekong Subregion (GMS) and recently in Africa [3].

The phenomenon of Antimalarial Drug Resistance (ADR) has led to the clinical retirement of Drug Classes 1–4 and is threatening the clinical efficacy of Drug Class 5-endoperoxides e.g. Artemisinins and their combinations. This is shown in the **Figure 1** below.

A critical look at **Figure 1** shows ADR as shown by the brown band generally causes a reduction in the clinical lifespan of the antimalarial drug as one moves interclass. This reduction in clinical lifespan has led to the retirement of drug classes 1–4 as first line treatment for malaria. Drug class 5; the Artemisinins and their combinations (ACTs) are currently under threat of resistance by the malaria parasite. ACTs are the WHO-recommended firstline and lastline (of some sorts) treatment for malaria. ACTs are one of the major factors responsible for the global successes recorded in the fight against malaria for about two decades now. The recent emergence of ACT-resistant strains of the malaria parasite in GMS and Africa has underlined the need for the discovery and development of novel therapeutic agents with novel mechanisms of action as one of the ways of tackling this hydra-headed monster. In tackling ADR, it is pertinent to zoom in on it and take a close look at it.

*Combating Antimalarial Drug Resistance: Recent Advances and Future Perspectives DOI: http://dx.doi.org/10.5772/intechopen.106507*

**Figure 1.**

*The impact of resistance on antimalarial drug classes. (adapted from a journal article, drawn with Microsoft excel).*

#### **2. What is antimalarial drug resistance (ADR)?**

Antimalarial Drug Resistance (ADR) can be best described as resistance to antimalarial drugs by the malaria parasite; Plasmodium species particularly *P. falciparum* which is responsible for the most deadly form of malaria - Falciparum malaria. *P. falciparum* is responsible for over 90% of the global malaria burden domiciled in Sub-Saharan Africa. It is the most virulent plasmodium species responsible for high morbidity and mortality rates of malaria observed in Africa particularly Sub-Saharan Africa. ADR has rendered most of the antimalarial drug classes clinically ineffective; these drug classes include 4-aminoquinolines, antifolates, mefloquine and atovaquone etc.

#### **2.1 Development of ADR**

When the malaria parasite is exposed to antimalarial drugs, initially it succumbs to the pharmacological action of the drug, over time develops resistance to it. Resistance development by the parasite is its adaptation to the new environment (with the presence of the drug) a complete departure from the old environment (absence of the drug). Continuous use of antimalarial drugs particularly at sub-therapeutic doses exposes them to the malaria parasite leading to the development of resistant strains of the parasite with several resistance phenotypes, some of these resistance genotypes might not necessarily have the resistance phenotypes. In Africa, the most frequent PfKelch13 mutation is A578S, this mutation does not confer Artemisinin resistance in vivo or in vitro [4]. The period between the first introduction of the antimalarial drug and the emergence of resistant strains of the parasite is laden with a variety of adaptive activities which may include genetic mutations which may include Gene Copy Number variations (CNV), Point mutations etc. An example of point mutation is the substitution of the Amino acid lysine with threonine at position 76 on the protein

(K76T). Gene copy number variants are deletions and amplification of a gene or a set of continuous genes and contribute to the great diversity of P.falciparum genome. *In vitro* studies have revealed their roles in parasite fitness phenotypes which include transmissibility, drug resistance, red cell invasion [5]. Resistance can also be imported into sensitive parasitic cells from neighboring resistant cells by R-plasmids transfer.

#### **2.2 Resistance phenotypes**

ADR by *P. falciparum* manifests with several resistance phenotypes which include; delayed parasite clearance, increased transmissibility, decreased schizont susceptibility, decreased gametocyte susceptibility, Ring stage resistance. Clinically, delayed parasite clearance is the phenotype used to establish resistance to antimalarial drugs. Parasite clearance rate can be used to measure delayed parasite clearance. It is quantified as the time taken by the antimalarial drug to reduce parasitaemia by half: for sensitive strains of *P. falciparum*, it is usually between 1 and 3 h, for resistant strains >5 h [6].

### **3. Mechanisms of drug resistance development**

ADR by *P. falciparum* occurs through various mechanisms which include;


#### **3.1 Factors affecting drug resistance development**

Having looked at the mechanisms of drug resistance, it is pertinent to also look at the enabling factors which include:

#### *3.1.1 Spontaneous mutations*

This occurs as a natural survival strategy of the parasite independent of the presence of the drug, Mutations occur *de novo*. The parasite's genome replication rate, *Combating Antimalarial Drug Resistance: Recent Advances and Future Perspectives DOI: http://dx.doi.org/10.5772/intechopen.106507*

#### **Figure 2.**

*Showing factors enabling resistance development and spread (adapted from www.malariasite.com with the aid of www.biorender.com).*

mutation rate per base pair per parasite generation are the principal determinants of spontaneous mutation [8].

#### *3.1.2 The antimalarial drug pharmacokinetics*

The selection of resistant mutants in the presence of the drug as shown in **Figure 2** is principally dependent on its pharmacokinetics (Slowly eliminated drugs with a long tail of sub-lethal dose generally select faster) and magnitude of drug use within the parasite population (the higher the drug pressure per parasite the faster the selection).

#### **4. Measures to tackle antimalarial drug resistance (ADR)**

ADR has retired drug classes 1–4 (**Figure 1**) clinically. Currently Drug class 5 (**Figure 1**) faces a big threat of being retired too by ADR. What measures has been taken to checkmate this hydra-headed monster and their limitations?

#### **4.1 Combination therapy**

The advent of chloroquine resistance led the World Health Organization (WHO) to approve some combination therapies which included various combinations of Antifolates e.g. Sulphadoxine 500 mg + Pyrimethamine 25 mg (SP), SP + Chloroquine (CQ), SP + Amodiaquine etc. The rationale for combination therapy is combining at least two drugs with different mechanisms of action against the malaria parasite. The aforementioned combination therapies are no longer in use as a result of resistance development to one or both drugs in the combination by the malaria parasite, secondly due to adverse effects of one or both drugs in the combination. With the introduction of Artemisinins as the mainstay for the treatment of uncomplicated *P. falciparum* malaria and the subsequent development of resistance to artemisinin

monotherapy, WHO approved Artemisinin-based Combination therapy (ACT) as the first line of treatment for uncomplicated and resistant *P. falciparum* malaria. The following ACTs approved by WHO are currently in clinical use;


The rationale for ACTs is combining a short-acting artemisinin with a long-acting partner drug whose duration of action provides the much-needed antimalarial cover long after the action of artemisinins has withered. This strategy was meant to overcome the phenomenon of ADR but sadly in 2009, there were reports of a deadly strain of *P. falciparum* (artemisinin-resistant P*. falciparum*) in the Greater Mekong Subregion (GMS) comprising Laos, Cambodia, Vietnam, Thailand, Myanmar and Yunnan Province in Southern China. The magnitude of this resistance threat in the GMS was to the extent of resistance to four out of the five WHO-approved ACTs for use in the region. This led to the setting up of the Regional Artemisinin-resistance Initiative (RAI) by the Global fund to address this emerging global health threat in the GMS in 2013. The outcomes of the RAI strategy in the GMS will be discussed under the section; Recent Advances against ADR.

#### **4.2 Continuous discovery of chemically and mechanistically novel antimalarial agents**

The need for unrelenting search for chemically and mechanistically novel antimalarial agents has been underscored by the growing threat of ACT-resistant malaria in the GMS. The past decade has seen an unprecedented renewed focus on the discovery of new antimalarial entities through extraordinary collaboration between academia (parasitologists, medicinal chemists, pharmacologists, clinicians) and industrial/private partnerships e.g. Medicines for Malaria Venture (MMV). The following promising antimalarial drug leads are products of such collaborations and are in the product development (patient exploratory) stages (**Figure 3**).

#### **4.3 Limitations**

Despite the above measures taken against ADR, limitations abound and they include development of resistance by *P. falciparum* to ACTs which presents as delayed parasite clearance. The huge cost involved in drug discovery and development projects is a great limitation to the search for novel antimalarial agents with novel mechanism(s) of action [9]. Some of the candidates have not gone beyond Phase II clinical trials because of safety concerns. The ones that crossed Phase II clinical trials do not

*Combating Antimalarial Drug Resistance: Recent Advances and Future Perspectives DOI: http://dx.doi.org/10.5772/intechopen.106507*

#### **Figure 3.**

*Showing progression of the clinical development of new antimalarial candidates over the past five years (source: www.researchgate.net, date accessed: 28th June, 2022).*

have significant antimalarial action due to resistance development and may exhibit unexplained loss of potency necessitating stoppage of such multi-billion dollar drug discovery and development projects.

#### **5. Recent advances**

Advances in the fight against ADR should among other challenges and areas of need address major limitations mentioned above. There has been renewed drive in terms of research to tackle head-on ADR using a multi-pronged approach, this has led to some recent advances which includes but not limited to the following:

#### **5.1 Regional artemisinin-resistance initiative (RAI)**

Launched in 2013 by the Global Fund to tackle Artemisinin resistance in the GMS (with the exception of Yunnan province in Southern China) [10] using a multipronged approach which included treatment and prophylactic strategies resulted in 88% reduction in indigenous malaria cases and 95% reduction in *P. falciparum* cases [1]. Partly responsible for the success story of malaria control in the GMS is the policy of Drug Rotation-exposure of the malaria parasite to different cycles of ACTs.

#### **5.2 Nanomimics**

This era of an emerging global threat (ACT-resistant malaria), an emerging, very promising strategy is the concept of nanomimics, an ingenious strategy developed by Najer et al. [11]. Researchers in Switzerland have successfully designed and tested host cell nanomimics. They developed a single procedure to produce polymer

#### **Figure 4.**

*Polymer-based RBC membrane nanomimics (source: Self-drawn with www.biorender.com).*

vesicles-small artificial bubbles with host cell receptors on the surface [11]. The concept of nanomimics is shown in **Figure 4**.

In **Figure 4**, an infected red blood cell undergoes various stages of the life cycle of the malaria parasite, mature schizonts rupture to release merozoites (light-blue) which are bound by nanomimics (nanoscaled polymer vesicles) preventing them from further invading normal red blood cells trapping them within the blood which exposes them to the immune system of the host.

Usually, the malaria parasites destroy their host cell after 48 h and then infect new red blood cells. At this stage they have to bind to specific cell receptors. Nanomimics as a result of their size and composition bind egressing parasites thus blocking the invasion of new cells [11]. The parasites are no longer able to invade cells and are consequently exposed to the host immune system, which kills them. Nanomimics exhibit a dual action; a therapeutic-like and vaccine-like effects. By preventing further invasion of red blood cells, it stops the progression of the disease (therapeutic-like action) and by exposing the bound merozoites to the host immune system for destruction (vaccine-like action) [12]. The intelligence of this strategy is that there is no payload (drug), malaria parasites are not exposed to any payload (exposure is key for resistance development). Non exposure of malaria parasites to drugs using this strategy could bring back exposure-caused, resistance-retired antimalarial drugs like chloroquine, SP, etc. as frontline antimalarial drugs [13]. This strategy is potentially resistance-proof and should be explored for possible translation into products for clinical trials.

#### **5.3 Targeted drug delivery**

This is one approach that inhibits resistance development by ensuring that adequate concentration of the antimalarial drug is delivered at the desired site of action ensuring rapid clearance of the parasite. The majority of antimalarial drugs under

#### *Combating Antimalarial Drug Resistance: Recent Advances and Future Perspectives DOI: http://dx.doi.org/10.5772/intechopen.106507*

development are lipophilic with poor plasma solubility and large biodistribution volumes which ultimately results in low accumulation in RBCs [9, 10]. This buttresses the need for targeted drug delivery to deliver optimum concentrations of drug within RBCs.

Targeted drug delivery using antibodies is a promising strategy that could be harnessed to combat resistant malaria. Antibodies have successfully been used to target pRBCs [14]. Antibody-targeted liposomes having on their surface Fð Þ *ab* <sup>2</sup> fragments of mouse monoclonal antibody raised against *P. berghei-*infected mouse erythrocytes significantly increased the therapeutic efficacy of chloroquine implying that target-specific liposomes can cure CQ-resistant malarial infections [15]. A tenfold increase in the therapeutic effect of CQ was observed when delivered in liposomes covalently functionalized with oriented, specific half antibodies against *P. falciparum* late form-infected RBCs [14]. Antibody-functionalized liposomes can discriminate pRBCs from non-infected RBCs specifically delivering antimalarial drugs to pRBCs in sufficient concentration to clear parasitaemia, however their use as targeting molecule in antimalarial therapy is limited by their high cost of production [16], high immunogenicity and the potential decrease in targeting efficiency due to variability in plasmodium protein expressed on the surface of pRBCs [17].

An interesting alternative to antibody-mediated targeted delivery is the use of certain glycosaminoglycans like heparin, heparan sulphate and chondroitin sulphate [18], which are found in the human body and are being recognized as one of the main pRBCbinding molecules [17]. Heparin bound to liposomes has a dual action as a pRBCtargeting molecule acts mainly on trophozoites of some pfEMP1-expressing lines [19] and on schizont stages [20] and as an antimalarial drug (Nanomimic polymer constructs) blocks the merozoites from invading red blood cells [21]. Heparin is cheaper than monoclonal antibodies resulting in heparin-bound liposomes having ten times less cost than immunoliposomes of similar targeting activity [18]. In addition, resistance to heparin as antimalarial drug have not been reported [21]. Heparin when covalently bound to liposomes has substantially reduced anticoagulant activity [18]. Heparin maybe limited in its application as a targeting ligand as a result of its anticoagulant actions but when used at non-anticoagulant concentration, it increased the efficacy of encapsulated primaquine threefold in *in vitro P. falciparum* cultures [18]. Heparin when compared with immunoliposomes for targeted drug delivery is cheaper, has lower or no immunogenicity and may potentiate the effect of the payload when used at non-anticoagulant concentrations. Heparin-related polysaccharides such as heparan sulphate, chondroitin sulphate can be used as targeting moieties, in comparison with heparin have much lower anticoagulant action [18].

#### **5.4 Triple artemisinin-based combination therapy (TACT)**

This is a combination of an artemisinin with two partner drugs as against the conventional one partner drug. This strategy is being proposed (in the face of the growing threat of artemisinin-resistant P.falciparum malaria which is causing delayed parasite clearance by ACTs) as a measure to tackle to artemisinin-resistant *P. falciparum* malaria. The result of a multicentre, open-label, randomized clinical trial of triple artemisinin-based combination therapy versus artemisinin-based combination therapy conducted in the GMS showed overall that 42-day Polymerase Chain Reaction (PCR) corrected efficacy of dihydroartemisinin-piperaquine plus mefloquine (97%; 95% CI 93–99) was higher than for dihydroartemisinin-piperaquine

(60%; 52–67) with a risk difference of 37%, 29–45; p<0.0001 [22]. There was no difference in efficacy between Artemether-Lumefantrine plus amodiaquine and Artemether-Lumefantrine-treated groups suggesting no relative advantage of having a triple combination of Artemether-Lumefantrine in treating resistant P.falciparum malaria [22]. Parasite half-lives in patients with Pfkelch 13 C580Y mutated infections were shorter in those treated with dihydroartemisinin-piperaquine plus mefloquine [mean = 6.93, SD = 1.77] than those treated with dihydroartemisinin-piperaquine [7.39 h (1.46); p = 0.019] [22]. This suggests albeit strongly that dihydroartemisininpiperaquine plus mefloquine as a promising TACT candidate against artemisininresistant *P. falciparum* malaria. Extensive clinical trials involving TACTs has to be done to really establish and justify a possible switch from ACTs to TACTs.

#### **5.5 Drug development**

There are promising drug candidates which are at various product development stages in researches conducted by some of the global pharmaceutical companies and some Research Institutions (Universities) or partnerships between the two resulting in drugs licensed for use with market authorization (refer to **Table 1**). The drug tafenoquine was recently licensed for radical cure of *P. vivax* malaria [23]. Medicines for Malaria Ventures (MMV) is at the forefront n novel drug discovery and development research.

#### **6. Future perspectives**

The way of the future is that of malaria eradication. The strategy to drive this should be anchored on the development of novel, smart, resistance-proof, antimalarial nanoformulations that with a single exposure lead to cure and prophylaxis (transmission-blocking ability). To this end, the application of nanotechnology in combating drug resistance in plasmodium species holds a lot of promises. Future strategies should target delivering antimalarial drugs via non-receptor-mediated pathways which are not under the genetic control of the parasite [24]. To this end, liposomal delivery holds a lot in stock since liposomes deliver drugs to their intracellular targets by fusing with the parasite's cell membrane. This technique can be used to deliver drugs whose resistance mechanisms are receptor-mediated e.g. chloroquine [25]. The parasite is less likely to modify the chemical composition of its cell membrane as that may affect its survival in terms of nutrient acquisition from its host. Future research should target what would be called a "Starving Strategy" whereby the parasite is starved of its nutrient supply from the red blood cell by developing Plasmodium surface anion channel blockers whose channel-blocking ability is by nanoadhesion - a phenomenon where nanocarriers block the nutrient pore channels by forming strong bonds with the negatively charged nutrient channels.

Nanomimics development and optimization is another smart move in beating the resistance trap of the parasite and should be explored [12]. Nanocarriers such as dendrimers present diverse opportunities of formulating combination drug products with novel mechanism of action that with a single exposure may possibly eradicate the parasite. Discovery and development of host-derived factors with therapeutic activity against the parasite are possible research options that will eradicate the parasite since the host-derived factors are not under the genetic control of the parasite.


*Combating Antimalarial Drug Resistance: Recent Advances and Future Perspectives DOI: http://dx.doi.org/10.5772/intechopen.106507*


 **1.** *Showing drug candidates in clinical development and their progression, accessed from www.mmv.org/research-development/mmv-supported-projects, date accessed: 29th June, 2022.*

#### *Malaria - Recent Advances and New Perspectives*

In all, nano-smart delivery of antimalarial drugs is the key to tackling and preventing resistance development in plasmodium species bringing global efforts steps closer to the actualization of the WHO ideal goal of malaria eradication by 2030.

### **7. Conclusion**

This chapter introduced malaria and Antimalarial Drug Resistance (ADR), its impact on global health, therapeutic interventions and their resistance by the malaria parasite. The mechanisms of resistance development by the parasite and factors causing it were discussed in detail. Armed with the knowledge of the foregoing, measures including nanotechnological approaches to combat the resistance trap of the parasite and their limitations were looked at and also the future perspectives in the fight against drug resistance by *P. falciparum.* It is envisaged that application of nanotechnology tools to develop antimalarial nanomedicines would help bring back antimalarial drugs that have been retired because of resistance of malaria parasites to these drugs and possibly lead to the development of novel, smart, resistance-proof formulations that will eliminate and possibly eradicate malaria globally.

#### **Conflict of interest**

The Authors declare no conflict of interest.

#### **Nomenclature**


### **Author details**

Samuel Uzondu\*, Adaeze Echezona, Chinekwu Nwagwu, Adaeze Onugwu, Lydia Ugorji, Chinazom Agbo, Franklin Kenechukwu, John Ogbonna, Paul Akpa, Petra Nnamani, Audu Momoh and Anthony Attama NanoMalaria Research Unit, Drug Delivery and Nanomedicines Research Laboratory, Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka, Nigeria

\*Address all correspondence to: samuel.uzondu@unn.edu.ng

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 11**

## Purine and Pyrimidine Pathways as Antimalarial Targets

*Yacoba V.T. Minnow and Vern L. Schramm*

#### **Abstract**

Malaria continues to plague the endemic regions of sub-Saharan Africa and Southeast Asia. With the current development of artemisinin resistance and a risk of failure of the current first line therapies, there is a growing need for novel antimalarials. Purine and pyrimidine metabolism in *Plasmodium* is distinctly different from the human host, making these pathways valid targets for the development of novel antimalarials. Targeting key enzymes in these pathways with transition state analogs has provided high affinity inhibitors. Transition state mimicry can also provide selectivity for the parasite enzymes over the homologous enzymes of the human host. Resistance of *Plasmodium* parasites to current antimalarials will be compared to resistance development induced by transition state analogs inhibitors, a feature that may contribute to decreased resistance development. Tight binding and specificity of transition state analog inhibitors provide important features for novel antimalaria therapy with low toxicity and prevention of antibiotic resistance.

**Keywords:** purine salvage, pyrimidines, antimalarials, transition state analog inhibitors, resistance

#### **1. Introduction**

Malaria remains the leading cause of mortality in the endemic regions of Sub-Saharan Africa and Southeast Asia. The WHO estimates that in 2020, there were 241 million malaria cases globally with 627,000 associated deaths, an increase from years prior [1, 2]. Additionally, although progress continues to be made in some regions, such as Southeast Asia reporting a 78% reduction in malaria cases, Sub-Saharan Africa accounts for more than 90% of the global malaria incidence. Children 5 years and younger are highly susceptible to malaria with a 77% mortality rate [1, 3, 4].

Malaria is caused by the apicomplexan parasite, *Plasmodium sp*. The five main species able to cause disease in humans are *Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium knowlesi* and *Plasmodium malariae. Plasmodium falciparum (P. falciparum)* is the most prevalent and the most lethal of these [5]. Malaria infection is established when an infected female anopheles mosquito takes a blood meal from humans and introduces sporozoites into the bloodstream which invade human liver hepatocytes. The sporozoites replicate in the hepatocytes for 5–8 days and can generate thousands of merozoites which are released to infect the erythrocytes [6, 7]. Asexual replication occurs in the erythrocytes where parasites

undergo several nuclear divisions and generate between 6 and 30 daughter merozoites per infected erythrocyte [6]. Symptomatic malaria is characterized by the sustained parasitization and destruction of red blood cells as well as the host immune responses. Thus, drug development efforts are directed toward the intra-erythrocytic asexual stage of the parasite.

Over the centuries-long battle with malaria, treatment has been developed from both natural product and chemical synthetic sources. Quinine, originally isolated from the bark of the cinchona tree, gave way to its synthetic counterparts, chloroquine, amodiaquine, and mefloquine, among others [8, 9]. Likewise, artemisinin was isolated from the Chinese herb, *Artemisia annua* (Qinghao), and gave way to the synthetic artemisinin derivatives now used in the first line malaria treatment, artemisinin-based combination therapy (ACT) [10]. However, resistance to these antimalaria treatments, have slowed global efforts toward the eradication of malaria [11–15]. Resistance development has led to increased efforts to identify novel antimalarials, including the recent development of the RTS,S malaria vaccine, providing a potential positive outlook for malaria control [16, 17]. New drug development efforts for malaria must identify novel targets, their mechanisms of action, and must be well tolerated with minimal side effects. Short treatment periods are essential to promote compliance and to minimize resistance development.

Transition state mimicry is a promising approach to malaria drug discovery. The technique utilizes features of transition state structure of essential target enzymes to produce tight binding and highly selective analogs [18]. Transition state analogs (TSAs) against the purine and pyrimidine pathway enzymes of *P. falciparum* have resulted in inhibitor molecules with the potential for new antimalarial combination therapies.

Rapid nucleic acid synthesis during intra-erythrocytic parasitic growth makes purine and pyrimidine synthesis in *Plasmodium* parasites an important target for novel drug development. Although both host and parasite share some enzymes in these pathways, key differences allow for *Plasmodium*-specific and selective molecules.

#### **2. Purine metabolism**

*Plasmodium* parasites are purine auxotrophs as they do not express the enzymes necessary to perform *de novo* purine synthesis [19, 20]. Substantial new DNA and RNA synthesis occurs during the asexual cycle of *Plasmodium*, requiring large amounts of purine and pyrimidine nucleotide precursors and the energetic contribution from ATP. Parasites therefore rely completely on the host erythrocytes for the salvage of purine nucleosides and nucleobases for the synthesis of adenylate and guanylate compounds. Hence the parasite genome contains highly expressed genes corresponding to purine transporters and purine salvage enzymes [21]. Human erythrocytes contain high (millimolar) concentrations of adenylate nucleotides, but *Plasmodium* parasites have no kinase to phosphorylate adenosine molecules or ribosyltransferases to salvage adenine from the host. Therefore, purine salvage in *Plasmodium* requires ATP catabolism, through hypoxanthine formation, by essential enzymes making up the purine salvage pathway (**Figure 1**) [22]. The central concept in targeting purine salvage pathways for *Plasmodium* involves prevention of hypoxanthine and inosine formation in both erythrocytes and parasites and/or prevention of hypoxanthine or inosine conversion to purine nucleotides in the parasites.

Purine precursors formed in erythrocytes must gain entry to the parasite for conversion to nucleotides. Pathways proposed to facilitate the uptake of purine

*Purine and Pyrimidine Pathways as Antimalarial Targets DOI: http://dx.doi.org/10.5772/intechopen.106468*

**Figure 1.**

*Purine salvage pathway in P. falciparum and human erythrocytes. In P. falciparum, but not in humans, there is a link to the polyamine pathway via MTA.*

nucleosides and nucleobases across the parasite membrane include; (1) saturable or facilitated transport of adenosine, (2) non-saturable, channel like transport and (3) the tubovesicular membranes induced by parasitization of the host erythrocytes [23, 24]. A major path for purine uptake is known to be the equilibrative nucleoside transporters (ENTs) that promote nucleoside and nucleobase transport into the parasite. Four classes of ENTs have been identified in *Plasmodium falciparum* and *Plasmodium vivax* (*Pf*ENT 1-4, *Pv*ENT 1-4) [24–28]. Inhibitors designed against *Pf*ENT1 prevent the uptake of purines and inhibit the growth of parasites in culture [29, 30]. The essentiality of the purine salvage pathway to the survival of *Plasmodium* parasites, makes targeting the component enzymes attractive for novel antimalarial therapies. These include adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) and hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT). Antimalarial validation of these targets has been confirmed by potent inhibitors that cause purine starvation and death of the parasites.

#### **2.1 Adenosine deaminase (ADA, EC 3.5.4.4)**

*Plasmodium falciparum* ADA (*Pf*ADA) catalyzes the zinc-dependent irreversible deamination of adenosine and deoxyadenosine to form inosine and deoxyinosine. *Pf*ADA can also catalyze the hydrolysis of 5′-methylthioadenosine (MTA) to 5′-methylthioinosine (MTI), a reaction that is distinctly absent from the mammalian ADA [31]. MTA is a product of the polyamine biosynthetic pathway, establishing that polyamine synthesis also plays a role in the purine salvage pathways for *Plasmodium* parasites. Parasites must metabolize MTA to prevent feedback inhibition of the polyamine biosynthetic pathway. Polyamine synthesis is also critical to the parasites since they cannot salvage polyamines from the host erythrocytes [see below]. Coformycin and 2′-deoxycoformycin are transition state or intermediate-like powerful inhibitors

#### **Figure 2.**

*Transition state analogs of HsADA and PfADA. The 5*′*-functionalized inhibitors provide improved selectivity for the parasite enzyme over the human enzyme. 5*′*-methylthio inhibitors are more potent thant the 5*′*-propylthio and 5*′*-phenylthio inhibitors.*

of ADA. They were originally identified as natural products and inhibit both human and parasite enzymes with picomolar affinities (**Figure 2**) [32]. 2′-Deoxycoformycin (Pentostatin) is an FDA-approved treatment for hairy cell leukemia, where the accumulation of adenosine or 2′-deoxyadenosine, leads to an unbalanced nucleotide pool, initiating apoptosis in B and T cells [32]. Unfortunately, there is insignificant inhibition of *Plasmodium* parasite growth by these inhibitors.

The dual specificity of *Pf*ADAs serves to guide the synthesis of novel *Pf*ADA inhibitors with a high degree of selectivity for the parasite enzyme. In addition, the transition state structures of *Pf*ADA and human ADA (*Hs*ADA) are similar but distinct, with *Pf*ADA displaying an early transition state and a more intermediate-like transition state for *Hs*ADA, corresponding to their distinct catalytic turnover numbers (*k*cat) (**Figure 3**). These differences are reflected by the state of protonation at N1 of the purine ring and the N1-H bond distances. The differences permit the design and synthesis of *Plasmodium*-specific transition state analogs [33]. Earlier work

*Purine and Pyrimidine Pathways as Antimalarial Targets DOI: http://dx.doi.org/10.5772/intechopen.106468*

#### **Figure 3.**

reported the synthesis of 5′-methythiolcoformycin (MT-coformycin), a sub nanomolar (*K*i\* = 0.43 nM) transition state analog inhibitor of *plasmodium* ADA with over a 20,000-fold selectivity over the human ADA. Other functionalized 5′- and 2′-deoxycoformycin molecules also retained selectivity for the *Plasmodium* enzyme (**Figure 2**) [31, 32]. MT-coformycin and coformycin inhibited the growth of *plasmodium* parasites in culture when MTA is used as the sole purine source, clearly demonstrating the deaminase as an essential step in conversion of MTA toward hypoxanthine [31, 34]. This effect is not observed when adenosine or MTI is used as a purine source, demonstrating that an intact polyamine pathway is not required for purine salvage in *Plasmodium* species. The high degree of selectivity of the 5′-functionalized coformycins demonstrates the importance of utilizing substrate specificity and transition state analysis to design target-selective inhibitors.

#### **2.2 Purine nucleoside phosphorylase (PNP, EC 2.4.2.1)**

The enzyme responsible for forming purine bases from nucleosides in the purine salvage pathway is PNP, catalyzing the reversible phosphorolysis of inosine to hypoxanthine and ribose 1-phosphate [35]. *Plasmodium* PNP substrate specificity includes MTI, to generate hypoxanthine and 5-methylthioribose 1-phosphate [36]. MTI appears to be a parasite specific metabolite and has not been identified in mammalian pathways [37]. Hypoxanthine serves as a key precursor to nucleotide synthesis; therefore, the parasite adopts multiple pathways to generate hypoxanthine. PNP also converts guanosine and 2′-deoxyguanosine to form guanine and (2-deoxy) ribose 1-phosphate (**Figure 1**). PNP displays specificity for 6-oxopurines with adenine containing purine rings having no effect as substrates or inhibitors [31].

*Plasmodium falciparum* PNP (*Pf*PNP) and human PNP (*Hs*PNP) have distinct structures and catalytic sites and share only ~20% sequence similarity [38]. Structurally, *Pf*PNP is more similar to PNP from bacterial sources and is homohexameric, displaying a trimer of dimer structure, while *Hs*PNP like other mammalian PNP is trimeric (**Figure 4**). Like *Pf*ADA, *Pf*PNP displays a broad substrate specificity that includes 6-oxopurine nucleosides and 5′-methylthio 6-oxopurine nucleosides as substrates. These differences form the basis for generation of species-specific inhibitors with discrimination for the *Plasmodium* enzyme.

The transition state structures of *Pf*PNP and *Hs*PNP, reveal similar catalytic mechanisms involving the formation of an oxocarbenium ion at the transition state (**Figure 5**). However, the two catalytic site interactions differ in the hydrogen bond and van der Waals interactions formed with the leaving group. The result is a more constrained active site environment at the transition state for *Pf*PNP than for *H*sPNP [39]. The

#### **Figure 4.**

*A, Homohexameric structure of PfPNP displaying a trimer of dimer conformation (1NW4). B, trimeric structure of HsPNP (1PF7).*

Immucillin-H (ImmH) transition state analog was designed to resemble the transition state structure of bovine PNP and inhibits that enzyme with a *K*i\* of 23 pM. Although there are distinct differences between the transition state structures of *Hs*PNP, *Pf*PNP and bovine PNP, ImmH is a potent inhibitor of all three enzymes, with *Hs*PNP *K*i\* = 56 pM and *Pf*PNP *K*i\* = 600 pM [40, 41]. DADMe-ImmH and DADMe-ImmG were developed as second generation PNP inhibitors designed to mimic the fully dissociative transition state, and thereby more closely resemble the transition state structures of *Hs*PNP and *Pf*PNP [42]. The introduction of a methylene bridge increases the bond distance between the 9-deazahypoxanthine (DADMe-ImmH) or the 9-deazaguanine (DADMe-ImmG) and the riboxocarbenium mimic. Additionally, the 9-deazapurine scaffold increases the p*K*a to permit protonation at N7 and the cation at N1′ mimics the cationic charge at this position of the transition state to more closely mimic the transition state. These features of the transition state translate to an improvement in the dissociation constant for *Hs*PNP from 56 pM for ImmH to 16 pM for DADMe-ImmH and to 2 pM for DADMe-ImmG. Transition state analogs bind tightly to their cognate enzymes by converting the catalytic potential into thermodynamic binding. The *k*cat for *Hs*PNP is approximately 50 times greater than for *Pf*PNP, and this is reflected in the affinity of the inhibitors for the two enzyme species. Thus, the dissociation constant (*K*i\*, the *K*d after slow onset inhibition) for *Pf*PNP is 500 pM for DADMe-ImmH and is 670 pM for DADMe-ImmG, reflecting its lower catalytic potential [42–44].

Inhibition of both *Hs*PNP and *Pf*PNP is required to cause a purine-less death of *Plasmodium falciparum* parasites in culture, as formation of hypoxanthine in erythrocytes or in the parasite will meet the needs for purine base salvage. In cell cultures, *Hs*PNP inhibition occurs at low concentrations of PNP inhibitors with inhibition of *Pf*PNP occurring at higher concentrations. Inhibition of both PNPs is thus required for the antiparasitic effects of the immucillins, with the IC50 values corresponding to the molar concentration of PNP present [45, 46]. The purine-less death induced by PNP inhibitors targets hypoxanthine production. Confirmation of hypoxanthine starvation as the mechanism of action, and the lack of other targets from this therapy, comes from the full restoration of parasite growth when hypoxanthine is added to culture media [45].

#### *Purine and Pyrimidine Pathways as Antimalarial Targets DOI: http://dx.doi.org/10.5772/intechopen.106468*

The genetic deficiency of *Hs*PNP is known to cause a T cell immune deficiency by causing accumulation of 2′-deoxyguanosine (dGuo), a metabolite completely dependent on *Hs*PNP for its recycling. Without *Hs*PNP, activated T cells transport dGuo and convert it to dGTP which can reach toxic levels for DNA polymerase. In humans, activated T cells are therefore sensitive to the loss of *Hs*PNP function with no effect observed in naive T cells, B cells, and other mammalian cells [47, 48]. The selective toxic effects of *Hs*PNP inhibition in human T cells is slow to develop, taking a year or more to be symptomatic in newborns. The more rapid effect in *Plasmodium* suggests that inhibiting both human and parasite PNPs for antimalaria therapy will have minimal side effects. Furthermore, oral administration of DADMe-ImmG clears *Plasmodium falciparum* parasites from infected *Aotus* monkeys [49]. Although not yet tested in clinical trials for malaria, the companion compound, DADMe-ImmH, is also a powerful inhibitor of both *Hs*- and *Pf*PNPs and has been in extensive phase 2 clinical trials for gout [50]. PNP inhibitors as an antimalarial therapy block hypoxanthine formation in both erythrocytes and parasites. Human blood hypoxanthine is present at approximately 1 μM, while *Aotus* blood was found to have approximately 40 μM hypoxanthine [49]. Therefore, PNP inhibition as an antimalarial therapy is expected to be a more effective in humans than in the *Aotus* test animal where several days of oral drug therapy were required to clear the parasites [49]. This hypothesis remains to be tested.

Other selective inhibitors remain an attractive option when developing novel antimalarial therapeutics. Therefore, using the dual specificity of *Pf*PNP for MTI, the transition state analog, 5′-methylthio-immucillin-H (MT-ImmH) was synthesized [46]. MT-ImmH has a 100-fold specificity for *Pf*PNP over *Hs*PNP, underscoring the importance of exploring transition state chemistry and substrate specificity to develop selective inhibitors (**Figure 5**) [46].

#### **Figure 5.**

*Reaction scheme and transition state structure of PNP (upper panel). Lower panel; transition state analogs of PNP with dissociation constants for HsPNP and PfPNP. Methylthio-Immucillin-H provides over 100-fold selectivity for the parasite enyzyme.*

#### **2.3 Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT, EC 2.4.2.8, EC 2.4.2.22)**

Hypoxanthine is the essential purine precursor for *Plasmodium* and HGXPRT is an essential enzyme in the *Plasmodium* purine salvage pathway, as the only enzyme to incorporate hypoxanthine into the parasite nucleotide pool. HGXPRT catalyzes the phosphoribosylation of hypoxanthine, guanine, and xanthine using 5-phosphoribosyl-α-D-1-pyrophosphate (PRPP) to generate inosine 5′-monophosphate (IMP), guanosine 5′-monophosphate (GMP) and xanthine 5′-monophosphate (XMP) respectively [51]. Pyrophosphate (PPi) is generated as the second product of the reaction (**Figure 6**). IMP serves as the precursor for the synthesis of both adenine and guanine-based nucleotides. The single distinction in substrate specificity between the *Plasmodium* HGXPRT (*Pf*HGPRT) and the human HGPRT (*Hs*HGPRT) is the inability of the human homolog to use xanthine as a substrate [52]. Xanthine in humans is converted to uric acid by xanthine oxidase and is the terminal end of purine metabolism, followed by its excretion into the urine [53, 54].

*Pf*HGXPRT has been identified as the most highly expressed purine salvage enzyme in *P. falciparum* and has been reported to have the highest activity compared to other enzymes of purine salvage identified by Reyes and coworkers [55]. Parasites may have evolved to use hypoxanthine as a key nucleotide precursor because it is the end-product of purine catabolism in human erythrocytes, with subsequent oxidation to uric acid occurring in other tissues. The high amounts of intracellular phosphates in erythrocytes favors the activity of PNP to produce hypoxanthine [55, 56]. Since its identification, *Pf*HGXPRT has been the most targeted enzyme for purine salvage in *P. falciparum*. Detailed kinetic and structural analyses have led to the design of many inhibitors of *Pf*HGXPRT activity. Challenges in targeting *Pf*HGXPRT include its similarities to *Hs*HGPRT and the need for the anionic phosphate group, making cell access a problem [57–60].

*Pf*HGXPRT and *Hs*HGPRT share 76% sequence similarity and 44% sequence identity [61]. Active site residues in contact with substrates and inhibitors are conserved (**Figure 7**). Purine nucleoside analogs and transition state analogs have been investigated and many prove to be good inhibitors of both *Pf*HGXPRT and *Hs*HGPRT.

#### **Figure 6.**

*Reaction scheme for HG(X)PRT. PfHGXPRT has a unique substrate specificity for xanthine. HsHGPRT does not.*

*Purine and Pyrimidine Pathways as Antimalarial Targets DOI: http://dx.doi.org/10.5772/intechopen.106468*

**Figure 7.**

*Two-dimensional ligand interaction map showing A, Immucillin-G phosphate bound to HsHGPRT (1BZY) and B, Immucillin-H phosphate bound PfHGXPRT (1CJB). Active side residues that make contact with inhibitors are similar in both enzymes.*

Acyclic nucleoside phosphonates (ANPs) however provide the greatest selectivity for the *Plasmodium* enzyme compared to phosphate-based compounds (**Figure 8**) [62, 63]. Crystal structures of *Hs*HGPRT in complex with ANP and free *Pf*HGXPRT suggest that the increased in selectivity when the phosphate groups are replaced with phosphonates is attributed to tighter hydrogen bonding around the phosphonate group and flexibility of the 6-oxo binding pocket [60].

TSAs are the tightest binding HG(X)PRT inhibitors reported to date. Although the first-generation transition state analogs, immucillin-G phosphate (ImmGP) and immucillin-H phosphate (ImmHP) were designed based on the proposed similarity of the transition state structure to other phosphoribosyltransferases, they bind potently but without discrimination for parasite or human HG(X)PRT (**Figure 8**) [41] and are subject to host enzymatic degradation by phosphomonoesterases. Structural analysis of TSAs bound to both *Hs*HGPRT and *Pf*HGXPRT as well as downfield proton chemical shift differences observed by NMR of bound inhibitors suggested that selectivity for *Pf*HGXPRT may be achieved by substituting TSAs with electron-withdrawing or electron-donating groups to alter hydrogen-bonding distances [41]. Recently, the transition state structure of *Pf*HGXPRT has been solved and provides detailed information on bond distances in the transition state [61]. With this knowledge, serinol-based mimics of the riboxocarbenium with a methylene bridge linking the oxocarbenium mimic to the 9-deazapurine ring were designed to mimic the bond distances in the transition state structure and synthesized. These compounds have proven to be the tightest and most selective inhibitors of *Pf*HGXPRT, with *K*i\* values as low as 650 pM and selectivity indices for *Pf*HGXPRT greater than 500-fold relative to *Hs*HGXPRT [64, 65].

HGXPRT TSAs are phosphate or phosphonate-based compounds with a net negative charge at physiological pH, making them impermeable to cell membranes. Prodrug approaches are therefore required for their biological activity. Prodrugs of *Pf*HGXPRT inhibitors involve ANPs with intramolecular esters in the form of lysophospholipid prodrugs, designed to be activated by intracellular phospholipase C,

#### **Figure 8.**

*First generation and second generation transtion state analogs for HG(X)PRT. Dissociation constants for HsHGPRT and PfHGXPRT are shown for each inhibitor. The second generation analogs provide greater selectivity for the PfHGXPRT. Lysophospholipid prodrug with biological activity against P. falciparum parasites in culture shown in lower panel.*

or phosphoramidate prodrugs with a multistep activation mechanism [66]. Prodrug approaches have been validated in several FDA-approved nucleoside antiviral drugs [67]. The prodrug approaches improve the biological activities of TSAs of *Pf*HGXPRT to give anti-parasite IC50 values in the micromolar range (2–7 μM) against *P. falciparum* parasites cultured in human blood (**Figure 8**) [65]. With a *K*i\* of 650 pM, disparity between the *K*i\* for the enzyme and the micromolar IC50 values of lysophospholipid prodrugs suggests that more efficient prodrug approaches are needed to fully capture the tight-binding capabilities of TSAs intracellularly. *Pf*HGXPRT is a challenging target because of the multiple cellular and membrane barriers between an oral drug and the intracellular parasites. However, similar challenges have been overcome for antivirals and they provide a lesson for prodrug approaches. In summary, *Pf*HGXPRT is a challenging but valid target for the development of novel antimalarials and TSAs currently provide the most promising approach.

#### **2.4 Downstream enzymes of purine salvage**

Given how essential purine salvage is to the survival of *Plasmodium* parasites, it is remarkable that the pathway evolved to rely on only three major enzymes, ADA, PNP and HGXPRT, based on the genome interpretation and protein expression levels in the parasites [55]. As concentration of free guanine in human blood is very low, it is not feasible for *Plasmodium* spp. to synthesize guanosine monophosphate (GMP) using HGXPRT [51]. Two enzymes downstream of HGXPRT, inosine 5′-monophosphate dehydrogenase (IMPDH) and GMP synthetase (GMPS) work sequentially to synthesize GMP (**Figure 1**) [68]. These two enzymes control GMP synthesis in most organisms [69]. IMPDH catalyzes the NAD<sup>+</sup> dependent reaction that coverts IMP to xanthine monophosphate (XMP) and NADH [70]. GMPS is composed of two domains, an ATP pyrophosphatase domain and a glutamine amidotransferase (GAT) domain [68, 71]. By this mechanism, GMPS catalyzes the ATP-dependent irreversible amination of XMP on carbon 2 to form GMP. Inhibitors of both IMPDH and GMPS have been shown to have antimalaria properties and have been the subject of recent drug development [21, 68].

Mycophenolic acid is an IMPDH inhibitor and inhibits *P. falciparum* parasites in culture with an IC50 of about 5 μM [21, 72, 73]. Bredinin is an inhibitor that has been shown to inhibit both IMPDH and GMPS [68, 74] and suggest that both IMPDH and GMPS are targets to explore for novel antimalarials. Although the IMP to GMP pathway involves phosphorylated intermediates, both mycophenolic acid and bredinin are non-phosphorylated natural products (or derivatives) that provide orally available access to tissues. Both mycophenolic acid and bredinin are used in immune suppression, and they have not been useful as antimalarials.

#### **2.5 Purine salvage and polyamine synthesis**

Polyamines are synthesized by the transfer of propyl amino groups from decarboxylated S-adenosylmethionine to putrescine to form spermidine (one transfer) and spermine (two transfers) [75]. MTA is the product of each transfer in the polyamine biosynthetic pathway. MTA is also a substrate for *Pf*ADA, whereby it is deaminated to methylthioinosine. These steps provide a path for this abundant product of the polyamine pathway to be channeled into the into the purine salvage pathway [76]. Polyamines are present at high amounts in the intraerythrocytic stages of *Plasmodium* spp., which can perform both *de novo* synthesis and salvage of polyamines [76, 77]. Given that parasites can only perform salvage of purines, the valuable purines used in the polyamine pathway are economically recycled. MTA is converted by the action of *Pf*ADA to produce MTI. MTI is a substrate for *Pf*PNP to form hypoxanthine and methylthioribose 1-phosphate. MTI is not found in human metabolism and recent studies have suggested that MTI production by *Plasmodium falciparum* in infected humans leads to activation of human Toll-like receptor 8 (TLR8), a signal for the stimulation of host innate immunity [37]. This connection provides an interesting link to the potential use of *Pf*PNP inhibitors as a potential therapeutic in human malaria. MTI is removed from *P. falciparum* metabolism exclusively by *Pf*PNP as *Hs*PNP does not use MTI as substrate. Therefore, inhibition of purine salvage via *Pf*PNP will increase MTI, increase signaling from TLR8 and have an immune stimulatory effect for the clearance of infected erythrocytes. Crosstalk between the polyamine pathway and purine salvage in *Plasmodium* parasites, together with the potential immunostimulatory effect of polyamine metabolites, underscores the importance of the polyamine pathway to purine salvage in *Plasmodium* parasites [37]. Inhibition of either the polyamine or the purine salvage pathways in *Plasmodium* parasites will be detrimental to parasites by; (1) purine starvation and death and (2) immune stimulation to promote clearance by host immune cells.

#### **3. Pyrimidine metabolism**

Opposite to the purine requirements of *Plasmodium falciparum*, the parasites are incapable of pyrimidine salvage from host erythrocytes; and pyrimidine synthesis requires the *de novo* pathway. This requirement contrasts with host cells with pathways for *de novo* synthesis and salvage of pyrimidine nucleotides [78]. Pyrimidine synthesis occurs by the action of six sequential enzymes to produce uridine monophosphate (UMP) as the primary product and a precursor to the other pyrimidine nucleotides (**Figure 9**) [79]. The enzymatic steps following UMP synthesis in *P. falciparum* are not well characterized, however, pyrimidine biosynthesis is linked to folate metabolism via the action of thymidylate synthetase (TS) [80]. Differences in the pathway between the human host and *Plasmodium* are important for selective targeting of *Plasmodium* parasites. In humans, the first three enzymes of the pyrimidine biosynthetic pathway, carbamoylphosphate synthetase II (CPS II), aspartate transcarbamoylase (ATC) and dihydroorotase (DHO) are a single protein, multiple domains, multifunctional enzyme complex, the CAD complex. In *Plasmodium*, CPS II, ATC and DHO are encoded as distinct genes, produce independent proteins and are organized on different locations on chromosomes, Ch. 13 for CPS II and ATC, and Ch. 14 for DHO [78, 81].

CPS II and ATC in *P. falciparum* are poorly characterized despite the role of CPS II as a key regulator of the pathway [20, 82]. The most thoroughly characterized enzymes of *Plasmodium* pyrimidine biosynthesis are dihydroorotate dehydrogenase (DHODH) and orotate phosphoribosyltransferase (OPRT) [83]. The transition state

#### **Figure 9.**

*De novo pyrimidine biosynthetic pathway. The pyrimidine biosynthetic pathway is conserved in Plasmodium and humans.*

structures of human (*Hs*OPRT) and *Plasmodium falciparum* OPRT (*Pf*OPRT) have previously been solved and transition state analog inhibitors of both *Hs*OPRT and *Pf*OPRT have been designed (detailed below).

#### **3.1 DHODH (EC 1.3.5.2)**

DHODH is the fourth enzyme in the pyrimidine biosynthetic pathway and is expressed as two isozymes. The isozyme expressed from chromosome 7 codes for a mitochondria-associated DHODH and chromosome 9 expresses a cytosolic DHODH, where the mitochondrial form is essential for *Plasmodium* growth [84]. DHODH catalyzes the formation of orotate from dihydroorotate, a rate-limiting step for pyrimidine biosynthesis. *Plasmodium* spp., like the human host, express a type II DHODH, localized in the inner membrane of the mitochondrion [84, 85]. The electron transport chain provides quinone for the redox activity of DHODH. Recent studies have highlighted *Pf*DHODH as a potential antimalaria target, showing that inhibiting *Pf*DHODH leads to parasite death in culture and animal models [85–87].

Selective inhibition of *Pf*DHODH has been achieved with DSM265, a triazolopyrimidine with a 4000-fold selectivity over mammalian DHODH. DSM265 has resulted in phase II clinical trials for the treatment of malaria, a first for any inhibitor of the parasite pyrimidine biosynthetic pathway. DSM265 inhibits both blood and liver stages of the malaria parasite with a biological efficacy similar to chloroquine in murine malaria models [87–89]. The pharmacokinetic analysis of DSM265 supported single dose therapy for use as a once weekly prophylactic [87]. Single dose administrations are considered important for patient compliance. Since the discovery of DSM265, DSM421, an improvement on DSM265 in terms of compound solubility, plasma stability and equal targeting of *P. falciparum* and *P. vivax* has also been reported in preclinical development [90]. Other studies have followed suit, using pharmacophore screening and structure-guided virtual studies to identify *plasmodium* specific DHODH inhibitors with submicromolar IC50 values [91, 92].

Pyrimidine biosynthesis is coupled to the mitochondrial electron transport chain (ETC) by DHODH and its requirements of quinones for catalytic activity (**Figure 9**). This coupling results in indirect inhibition of DHODH by ETC inhibitors such as atovaquone, an approved antimalarial often used in combination with proguanil [93]. Although DHODH and the linked mitochondrial ETC were promising targets with extensive discovery and development programs providing powerful inhibitors, the rapid development of resistance in clinical trials has hindered their development toward approved drugs [94, 95].

#### **3.2 OPRT (EC 2.4.2.10)**

Reduction of dihydroorotate by DHODH produces orotic acid and the following step, catalyzed by *Pf*OPRT, catalyzes the production of the nucleotide orotidine monophosphate (OMP) from orotate and PRPP. OMP is subsequently converted to UMP by OMP decarboxylase (OMPDC). *Plasmodium* and human OPRTs differ in the early steps of the pyrimidine biosynthetic pathway. Thus, human OPRT is fused to OMPDC to form the single protein, bifunctional enzyme called UMP synthase [96]. In *Plasmodium*, OPRT and OPMDC have been purified as monofunctional enzymes however, there are recent reports suggesting that OPRT and OPMDC may exist as a heterotetrameric enzyme in *Plasmodium* with the potential for hydrolysis into the single enzymes during purification [83, 97, 98]. Detailed kinetic characterizations of

both human and *P. falciparum* OPRTs (*Hs*OPRT and *Pf*OPRT respectively) have been reported and active site differences between the homologs have been characterized [83, 98].

The essential function of OPRT in pyrimidine biosynthesis and therefore for parasite proliferation has been demonstrated by the selective killing of cultured *P. falciparum* parasites by pyrazofurin, an inhibitor of OPRT with anti-parasite IC50 values of 6–20 μM [21, 99]. Additionally, 5-fluororotate and 5-aminouracil have inhibitory activity against *Pf*OPRT with IC50 values of 42 nM and 8 μM respectively [21]. 5-Fluororotate selectively targets *P. falciparum* parasites in culture with an IC50 of 6 nM [100]. The mechanism of action is proposed to arise from an indirect effect of the toxic 5-fluorodeoxyuridylate metabolite on TS [101]. *Plasmodium* express TS as a single protein, multi-domain bifunctional enzyme with dihydrofolate reductase (DHFR) whereas TS exists as a monofunctional enzyme in mammals [21, 99, 100].

Transition state analysis of *Pf*OPRT and *Hs*OPRT reported an SN1 transition state with a partial dissociative orotate and riboxocarbenium ion character for both enzymes [102]. Despite differences in the enzyme organization, *Pf*OPRT and *Hs*OPRT possess similar transition state structures, where the ribocation is fully developed, the dianionic orotate is fully dissociated and there is weak participation of the pyrophosphate nucleophile (**Figure 10**) [102]. The transition state structures provided information for the design of several TSAs with nanomolar affinities for both *Hs*OPRT and *Pf*OPRT (**Figure 10**) [103]. Inhibition of *Hs*OPRT might have potential utility for the treatment of autoimmune diseases and some malignant neoplastic diseases. However, no anti-parasitic activity was observed for any of the TSAs in cultured *P. falciparum.* The lack of activity was attributed to permeability barriers and target

#### **Figure 10.**

*Upper panel: OPRT reaction scheme and transition state structure. The transition state structure is similar for PfOPRT and HsOPRT. It features a fully developed ribocation and a fully dissociated dianionic orotate with weak participation of the pyrophosphate. Lower panel: Transition state analogs synthesized and tested on PfOPRT and HsOPRT.*

access, but no permeability studies have been reported [103]. *Pf*OPRT remains a viable antimalarial target requiring further studies to elucidate and develop speciesspecific antimalarials.

#### **3.3 OMPDC (EC 4.1.1.23)**

The decarboxylation of OMP to form UMP is catalyzed by OMPDC, the sixth enzyme in the *de novo* pyrimidine biosynthetic pathway [78]. OMPDC has achieved catalytic notoriety as it has been considered the most proficient pure protein catalyst, giving a catalytic reaction rate enhancement of approximately 1017 [104, 105]. Unlike other decarboxylases, OMPDC requires no metal ions or cofactors for its catalytic activity [105]. Recent studies indicate that *Plasmodium falciparum* OMPDC may form a tight heterotetrameric complex with OPRT (*Pf*OMPDC2-*Pf*OPRT2) with properties distinct from the bifunctional human OPRT [106, 107]. Although both are expressed and encoded by two separate genes, unique amino acid insertions in both proteins not present in the homologous proteins of other organisms allow the formation of the heterotetrameric complex [98, 106]. Additional support comes from the kinetic characterization of fused *P. falciparum* OPMDC and OPRT, expressed in *Escherichia coli*, where the catalytic efficiency of the fused enzymes was enhanced several orders of magnitude relative to either enzyme acting as monofunctional proteins, leading the authors to call the fusion a 'super' enzyme [97].

Nucleoside 5′-monophosphate analogs, 6-azauridine 5′-monophosphate (AzaUMP), allopurinol-3-riboside 5′-monophosphate (allopurinol MP), pyrazofurin 5′-monophosphate (pyrazofuin MP), and xanthosine 5′-monophosphate (XMP) are potent inhibitors of both human and parasite OMPDCs (**Figure 11**) [108]. These display a strong preference for *Pf*OMPDC, with selectivity ranging from 11 to 150-fold preference for the parasite enzyme [108]. The differential binding of XMP has been investigated by comparing X-ray crystal structures of both enzymes. Few differences in active site residues were observed and specificity may be as a result in hydrogen bonding differences between ligand and active site residues (**Figure 12**). Another reported difference is the βα5-loop present at the dimerization interface, which displays different conformations and amino acid substitutions in *Pf*OMPDC compared to *Hs*OMPDC and is postulated to cause different active site rearrangements around the pyrimidine binding region [108]. The phosphate binding loop is also larger in *Hs*OMPDC than in *Pf*OMPDC [108]. Repositioning of the loop is a proposed requirement to bind XMP as observed for XMP-bound *Pyrococcus horikoshii* OMPDC [108]. Interestingly, the phosphate loop is the same size and shape in *P. horikoshii* OMPDC and *Pf*OMPDC, therefore, these enzymes may bind XMP similarly. XMP is a 150-fold more potent inhibitor of *Pf*OMPDC than of *Hs*OMPDC [108].

No crystal structures of inhibitor bound *Pf*OMPDC are available to understand the basis for inhibitor selectivity. However, the high preference of inhibitors for parasite OMPDC compared to the human enzyme indicates the possibility of developing potent and selective antimalarials. Inhibition of OMPDC has focused on nucleotide analogs, where the 5′-phosphate is a significant contributor to binding. A substantial barrier to drug development for nucleotide inhibitors is the anionic charge, as phosphoryl anions are membrane impermeable and phosphomonoesters are susceptible to phosphomonoesterases. Prodrug approaches, now being used in antiviral therapy for the delivery of phosphate nucleosides may be useful in development of OMPDC inhibitors.

*Nucleoside 5*′*-analogs of OPMDC showing inhibitory constants for both PfOMPDC and HsOMPDC. Inhibitors are highly selective, 11–150-fold for PfOMPDC over HsOMPDC. Values are as reported in [108].*

#### **Figure 12.**

*2-dimensional ligand interaction map of UMP bound A, PfOPMDC (6DSR) and B, HsOMPDC (2V30). Similar active site residues make contact with UMP.*

#### **3.4 DHFR (EC 1.5.1.3) and TS (EC 2.1.1.45)**

*De novo* purine synthesis in the host and pyrimidine synthesis in both host and parasites are dependent on folate metabolites for the donation of carbon units. The pyrimidine biosynthetic pathway is linked to folate metabolism by TS, which requires 5,10-methylene tetrahydrofolate as a methyl donor for its catalytic activity [109]. DHFR and TS are both targets for human cancers [110]. In *Plasmodium falciparum*, DHFR and TS are expressed as a single protein, two-domain bifunctional DHFR-TS enzyme system, catalyzing the synthesis of deoxythymidylate monophosphate (dTMP) from dUMP and the NADPHdependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) [80]. The bifunctional coupling of adjacent steps in the pathway is proposed to cause "substrate channeling", an efficient utilization of folate production for the synthesis of purines, pyrimidines, and amino acids [111, 112]. Since malaria parasites are unable to salvage pyrimidines, if metabolites or cofactors required by the folate pathway are cut off, thymidine biosynthesis is blocked, and parasites die from pyrimidine starvation [20, 80]. Thus, DHFR has been a frequently-drugged target for antimalaria therapy.

Pyrimethamine (PYR) and proguanil (PG) are approved antimalaria therapeutics which selectively target *Pf*DHFR by several hundred-fold compared to the human counterparts [20, 113]. Mutations in the *DHFR* gene conferring resistance to both PYR and PG have led to diminished clinical effectiveness as single therapeutics for malaria. However, the combination of pyrimethamine with sulfadoxine (SP), which blocks dihydropteroate synthase, a folate precursor, act synergistically in the treatment of severe malaria [114]. Despite the prevalence of molecular mutations causing resistance to SP, WHO still recommends SP for intermittent preventative treatment for pregnant women and, often, in combination with amodiaquine, an inhibitor of *Plasmodium* heme polymerase, for seasonal malaria prevention [115]. PG is also used in combination with atovaquone, an inhibitor of the mitochondrion electron transfer chain, which also directly impacts pyrimidine biosynthesis because of its coupling to DHODH [116, 117].

Recent searches for additional inhibitors of DHFR-TS identified the antimicrobial triclosan to inhibit the proliferation of intraerythrocytic *P. falciparum* and is a 775 nM inhibitor of *P. vivax* DHFR. Triclosan displays a 20-fold selectivity for *Plasmodium* enzymes relative to human DHFR [118]. To preempt the development of resistance, Tarnchompoo and coworkers have developed hybrid inhibitors of DHFR with both flexible and rigid side chains that target both wild-type and multiple resistance *P. falciparum* [119]. Hybrid inhibitors include sub-nanomolar inhibitors of *Pf*DHFR with 10-fold selectivity over *Hs*DHFR [119]. Flexible cycloguanil analogs have been characterized that are low nanomolar inhibitors of multiple mutant *Pf*DHFR [120]. Fragment-based screening approaches to inhibitor design have identified nonpyrimidine scaffolds that inhibit *Pf*DHFR-TS with IC50 in the range of 28–695 μM and are highly selective over *Hs*DHFR [121]. This approach offers a new avenue to tackle antifolate resistance and develop new antifolate antimalarials, but remains in development [121].

DHFR-TS is a validated target for antimalaria combination therapy by blocking synthesis of dTMP. The functional characteristics of DHFR-TS including its crystal structure and the structures of drug-resistant DHFR-TS allows for the identification and development of next-generation selective inhibitors of either catalytic domain that will address resistance to current antifolate drugs for malaria. New approaches

are being explored, including peptide-based antagonists of *Pf*DHFR-TS in the quest for novel and potent antifolate/anti-pyrimidine drugs [122].

#### **4. Transition state analogs and drug development**

Transition state theory postulates that chemically stable mimics of the enzymatic transition state will bind tightly to the target [123, 124]. All chemical, including enzymatic reactions, proceed via a transition state, a transient high energy species that lies along the reaction coordinate between reactants and products [125, 126]. Transition states are the balance point of a catalytic reaction where bonds are partially broken or formed and the probability for product formation and return to reactants is equal. Enzymes have evolved to highly favor the transition state geometry, hence their very large rate enhancement factors, typically of 1010 to 1015, a very large decrease in the activation energy of the reaction [18]. Chemically stable mimics of the transition state bind to and stabilize this favored enzymatic transition state geometry, capture part of the transition state energy and bind very tightly [18, 127–129]. Therefore, transition state analysis provides a powerful tool to develop molecules with high inhibitory potential for their target enzymes. Kinetic isotope effects (KIE) remain the best approach to study enzymatic transition states and together with quantum computational chemistry, allows the construction of electrostatic potential maps that provide information on the transition state structure [18]. This information enables the design of transition state analogs that bind with high fidelity to their cognate enzymes, millions of times tighter than substrates. Transition state analogs are some of the tightest binding enzymatic inhibitors and have the propensity to bind their target enzymes with dissociation constants in the nanomolar to femtomolar range (10−9 to 10−15 M) [123, 125, 126]. A feature of transition state analogs is the ability to convert the enzyme potential for catalysis into binding energy, therefore the more catalytically efficient the enzyme, the tighter the potential for transition state analog binding [127].

The immucillins are chemically stable transition state analogs that mimic the ribocation transition state of N-ribosyltransferases, enzymes that include *Pf*PNP and *Pf*HGXPRT, both important in purine salvage in *P. falciparum* [50]. Immucillin-H (also known as: BCX1777, Forodesine and Mundesine) (**Figure 5**) is an inhibitor of PNPs and has been approved in Japan for the treatment of resistant or relapsed peripheral T cell lymphoma (PTCL). Immucillin-A, as Galidesivir (also known as BCX4430) continues in phase I - II clinical trials for antiviral therapy. It is converted to the triphosphate form where it blocks RNA chain elongation in RNA viruses, including Yellow Fever and SARS-CoV2 [130]. DADMe-Immucillin-H (also known as: BCX4208, Ulodesine) has completed phase II clinical trials for gout, by virtue of its powerful inhibition of human PNP, an essential step in formation of uric acid in humans [50]. No immucillins or related transition state analogs for purine and pyrimidine pathways have yet entered clinical trials for antimalaria therapy though several are in preclinical testing [128].

The immucillins differ from traditional antibiotic discovery. Antibacterials are often discovered by cell wall screening or genomic targeting to inhibit cell wall synthesis [131]. Immucillins and related transition state analogs are designed to mimic the geometry of the transition state of their specific enzymes [129]. The resulting transition state analogs can be powerful tight-binding inhibitors with exquisite specificity for their targeted enzymes. Transition state inhibitor design is adding new candidates for drug development.

#### *Purine and Pyrimidine Pathways as Antimalarial Targets DOI: http://dx.doi.org/10.5772/intechopen.106468*

Immucillins in preclinical development as antimalaria drugs include the PNPtargeting DADMe-ImmG, a powerful inhibitor that is a picomolar inhibitor for both human and *Pf*PNPs. Administration at nanomolar concentrations to parasites cultured in human erythrocytes caused purine-less death of *P. falciparum*. Testing in *Aotus* primates infected with *P. falciparum* at an oral dose of 50 mg/kg and dosed for 7 days gave robust parasite clearance and no parasitemia was detected by day 6 of treatment, along with complete inhibition of host and parasite PNP [49]. Recrudescent parasites appeared several days after treatment stopped [49]. *Aotus* monkeys, however, have approximately 40 μM circulating hypoxanthine, compared to approximately 1 μM in humans [49]. Therefore, clearing *P. falciparum* from *Aotus* is a more stringent test for the antimalaria efficacy of DADMe-ImmG than anticipated in human infections. DADMe-ImmH, the compound tested extensively in phase I and phase II clinical trials for gout, has a good safety profile in humans. DADMe-ImmH has an inhibitory potential for both *Hs*PNP and *Pf*PNPs similar to DADMe-ImmG and is a strong candidate to enter human clinical trials [50]. The efficacy of DADMe-ImmH has been established in clinical trials. A single oral dose of 0.5 mg/kg completely inhibits erythrocyte PNP with inhibition lasting for 120 days, the lifetime of the erythrocyte, making it a candidate for single-dose therapy of *P. falciparum* [132]. As inhibition of both human PNP and *Pf*PNP is required for antimalarial properties, a single dose therapy has potential as a novel antimalarial, and as described below, has a reduced potential for the induction of resistance mutations.

#### **4.1 Antimalaria resistance to transition state analogs**

*P. falciparum* has developed resistance to most approved antimalarials, contributing to the difficulty of disease-eradication efforts worldwide. Rapid development of resistance has led to the clinical failure of several potent antimalaria drugs early in clinical trials and has resulted in World Health Organization recommendations for combination drug therapy for malaria treatment. Chloroquine which was once the gold standard treatment for malaria was widely used until the 1950s, when widespread resistance prompted its removal from the list of approved malaria therapeutics [133, 134]. Resistance soon followed for drugs such as atovaquone, pyrimethamine, proguanil, cycloguanil, sulfadoxine and sulfadoxine-pyrimethamine (SP) [133, 134]. Recently, resistance to artemisinin, one partner of the current first line treatment has been reported in the endemic regions of Sub-Saharan Africa and the Greater Mekong region of Southeast Asia [12, 14, 135, 136].

In the field, drug resistance is characterized by delayed-clearance of parasites from the blood of infected individuals [11]. Resistant parasites often have point mutations and gene amplification which can result in decreased drug uptake, increased efflux of drugs, target overexpression, target modification that reduces drug binding, and inactivation of a drug by modification (**Figure 13**) [134]. Knowledge of these mechanisms to resistance have prompted the search for novel and unique antimalaria compounds followed by investigation of the rates and mechanisms of resistance by subjecting cultured *P. falciparum* parasites to constant pressure of the novel compounds.

One such study involving DSM265, the *Pf*DHODH inhibitor. Using a drug pressure of 3 × EC50 revealed a minimum inoculum for resistance (MIR) of 2 × 106 compared to 2 × 107 for atovaquone [87]. Selection of resistant parasites to DSM265 required 8 x EC50 of DSM265 compared to 33 × EC50 required to suppress atovaquone resistant parasites. The resistance mechanism in Dd2 clones (resistant to chloroquine,

#### **Figure 13.**

*Antimalarial resistance mechanism frequently employed by Plasmodium parasites to overcome drug pressure.*

pyrimethamine and mefloquine) revealed both gene amplification of the DHODH gene and the G181C mutation that confers a 13-fold shift in IC50 of the drug compared to wild-type enzyme and 26-fold reduction of the EC50 in parasite growth assay. Kinetic characterization of the recombinant mutant enzyme revealed a 2-fold increase in the *k*cat, and *K*m was unchanged [87]. Resistance to DSM265 in Dd2 parasites developed rapidly with a profile similar to resistance development for atovaquone. However, resistance was not as easily generated at higher concentrations of DSM265 in other *P. falciparum* strains, questioning if resistance would develop in the field [90]. DSM265 in phase II clinical trials for malaria led to the appearance of resistance during the clinical trial phase [94].

In contrast to DSM265, resistance to transition state analogs is not easily generated. Loss of transition state features in the protein also generates severe catalytic penalties predicted to induce a fitness cost to the parasites. For example, treatment of cultured *P. falciparum* with DADMe-ImmG for 1 year in culture resulted in a 6-fold resistance (6-fold increase in IC50) [44]. The resistance was caused by a 6-fold amplification of wild-type *PfPNP* gene and protein levels. After 3 years of drug pressure (over 2136 clonal selections) in cultured cells, increased resistance was marked by a 12-fold amplification in the target *PfPNP* gene [44]. The 12-fold gene amplification was accompanied by two point mutations in PNP occurring in separate clones (M183L and V181D) to give rise to a 500-fold increase in the IC50. Interestingly, point mutations were only present in 50% of the transcripts and the remainders were wild type [44]. Recombinant expression of mutant PNP revealed that the kinetic properties were incompatible with the purine salvage function of PNP [44]. Particularly, the M183L mutation resulted in a 17,000-fold decrease in the catalytic efficiency (30-fold decrease in *k*cat) of the enzyme and a 39,000-fold decrease in affinity for DADMe-ImmG [44]. Therefore, the mutation reduced the catalytic efficiency and DADMe-ImmG efficacy by approximately the same amount, highlighting a key feature of transition state analogs. Mutations that prevent binding of the analog are expected to decrease catalytic activity to the same degree.

Resistance to DADMe-ImmG in *P. falciparum* arises by a unique mechanism. Hybrid PNP expression of 50% mutant M183L subunits and 50% wild-type subunits is proposed to generate hybrid hexameric PNPs demonstrating a 6-fold decrease in

#### *Purine and Pyrimidine Pathways as Antimalarial Targets DOI: http://dx.doi.org/10.5772/intechopen.106468*

catalytic efficiency, as three of the six subunits are native. This hybrid hexameric construct of three native and three mutant subunits displays negative cooperativity in binding to DADMe-ImmG, always leaving a fraction of the native subunits free to catalyze the formation of hypoxanthine [137]. Therefore, in addition to 12-fold target overexpression and point mutations, *P. falciparum* also employs hybrid multimeric PNP to achieve robust resistance [137]. This example emphasizes the severe catalytic costs and threat to biological function that resistance to transition state analogs produces in the parasites. This slow development of resistance to DADMe-ImmG suggests that resistance may be slow to develop in the field.

Conditions for generating laboratory DADMe-ImmG-resistant *P. falciparum* clones are stringent, requiring up to 3 years of drug pressure *on the same culture* before robust resistance appeared. In the field, effective anti-parasitics clear parasites in a matter of days, and it is unlikely that parasites would encounter conditions of continuous drug pressure. Notwithstanding, these *in vitro* resistance assays help identify mechanisms and molecular markers of resistance and resistance phenotypes that will aid in recognizing resistance development in the field. Like most antimalarials, the proposed use of DADMe-ImmG or related transition state analogs would be in drug combinations to provide an extra layer of protection against development of resistance.

#### **5. Summary and conclusion**

During the intraerythrocytic stages, *Plasmodium* parasites proliferate rapidly and require extensive nucleic acid synthesis. The building blocks are either salvaged from the human host in the form of purines or synthesized de novo (pyrimidines). There are distinct differences between the purine and pyrimidine synthesis pathways of humans and the malaria parasites. Therefore, targeting these pathways can aid in the development of novel chemotherapeutic agents to combat emerging resistance to the current first line antimalarials. Transition state analogs against various purine and pyrimidine pathway enzymes are emerging as promising candidates for antimalarial therapy. The slow development of resistance and the unusual resistance mechanism employed by the parasites to drug pressure against transition state analogs may lead to slow resistance development in the field. Finally, the recent advances made with the RTS,S vaccine in endemic African countries is promising and point in the right direction for population control of malaria. However, until an effective vaccine is approved for widespread use, chemotherapeutic agents managing the disease and vector control measures are still needed.

#### **Acknowledgements**

Preparation of this manuscript was supported by NIH research grants (AI127807 and GM041916). We thank our collaborators in Dr. Peter C. Tyler's group at Ferrier Research Institute, Victoria University of Wellington, New Zealand, for the synthesis of the Immucillins and transition state analogs.

#### **Conflict of interest**

The authors declare no conflict of interest.

*Malaria - Recent Advances and New Perspectives*

#### **Author details**

Yacoba V.T. Minnow and Vern L. Schramm\* Department of Biochemistry, Albert Einstein College of Medicine, New York, USA

\*Address all correspondence to: vern.schramm@einsteinmed.edu

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 12**

## Antioxidant Efficacy of Selected Plant Extracts Debilitates the *Plasmodium* Invasion through Erythrocytic Membrane Stabilisation - An *In Vitro* Study

*Urja Joshi, Dhara Jani, Linz-Bouy George and Hyacinth Highland*

#### **Abstract**

Most dangerous and prevalent form of malaria is caused by the *Plasmodium falciparum* mediated malaria and poses the greatest threat to the humans. Emergence of multi drug resistant parasite hindered the prevention of malaria burden worldwide. This study is mainly focused on the erythrocytic membrane stabilisation using regionally available medicinal plant extracts and its corelation with the oxidative stress generated during the intracellular erythrocytic stages development of *Plasmodia*. The results disclosed that antioxidant potential of the medicinal plants can diminish the reactive oxygen species generation leads to restrict the plasmodial invasion into erythrocytes ultimately decreases the parasitic load. Hence, the evidence of the effective phytochemicals present in the selected medicinal plants can be the promising anti-plasmodial drug candidates as a future perspective.

**Keywords:** *Plasmodium falciparum*, erythrocytic membrane stabilisation, antioxidant activity, anti-plasmodial activity, oxidative stress

#### **1. Introduction**

**221** The burden of malaria is still felt worldwide and caused by Apicomplexa parasite *Plasmodium* spp., in which, *Plasmodium falciparum (P. falciparum*) primarily causes severe malaria and remains the leading cause of morbidity and mortality worldwide. According to World Health Organisation (WHO), 241 million cases of malaria and 627,000 deaths were reported globally in 2022 [1] in which the WHO African Region has consistently reported a significant portion of the worldwide malaria burden. Moreover, Malaria cases in the WHO South-East Asia Region dropped significantly and made up roughly 2% (5 million cases in 2020) of all malaria cases worldwide [1]. Still, more than 4,00,000 people around the world killed due to malaria every year [2, 3]. It mostly occurs due to the delay in treating *P. falciparum* caused malaria.

*Plasmodia* requires at least two hosts a) Female *Anopheles* mosquitoes – for sexual cycle b) Human beings – for asexual cycle, to accomplish its life cycle. It infects in form of sporozoites via female *Anopheles* mosquitoes' bites enters the human being, within 60 mins it invades hepatocytes via circulatory system [4]. Eventually sporozoites matures and complete its pre erythrocytic stages into 6–15 days, which is clinically remains silent, and leave the liver in form of merozoites and enters the circulation to invade erythrocytes [4]. During erythrocytic stages, merozoites develop into (trophozoites – ring stages – schizonts - merozoites) in <48 hrs and on the time of maturation thousand of merozoites leave that erythrocyte to infect new erythrocytes. Bursting of erythrocytes into circulation onset the progression of symptoms including chills, fever, headache, etc. associated with malaria [5]. Severe malaria includes the series of complications include cytoadherence, and sequestration found in *Plasmodium falciparum* infected erythrocytes (*Pf-*iEs) leads to unregulated inflammatory processes, sequestration, coma, severe anaemia, multiple organ dysfunctionality and cerebral malaria like complications in vital organs [5]. In addition, mortality rates in *P. falciparum* caused malaria remained elevated mainly in pregnant women, young children >5 years, etc. [6]. Although the availability of wide spectrum of anti-malarial drugs in the markets, emergence of drug resistant of malaria parasite especially of *P. falciparum* has created an urgent demand for newer, more efficacious, anti-plasmodial agents, with minimal side effects. The search for a safe, efficient treatment agent for the control and management of this dreadful disease may perhaps now have an answer thanks to several plant extracts and formulations that are highly concentrated in powerful phytochemicals. According to estimates from the WHO, 80% of people rely on herbal remedies as their primary form of healthcare. Around 21,000 plant species have the potential to be used as medical plants, according to the WHO [7]. The prophylactic anti-malarial medication derived from medicinal plants is one possible source since certain secondary plant compounds have a significant potential for cell-cell and cell-molecular interactions [8]. Natural products have been essential in the discovery and development of new medications for a long time since plant primary and secondary metabolites have significant biological functions [9].

Here, two medicinal plants:


#### *Antioxidant Efficacy of Selected Plant Extracts Debilitates the* Plasmodium *Invasion… DOI: http://dx.doi.org/10.5772/intechopen.106844*

habitats where rainfall is restricted to 150 to 1000 mm, as well it found in typical habitats including roadside sand dunes, seashore dunes, and heavily populated urban areas [14]. *C. procera* possess a wide range of pharmacological activity. Plant part such as root bark, stem bark, leaf, flower, and latex and their extracts, fraction, and isolated compound proved larvicidal, anticancer, acaricidal, schizonticidal, antibacterial, anthelmintic, insecticidal, anti-inflammatory and antidiarrheal special effects [15, 16]. Numerous cardenolides, alkaloids, flavonoids, sterols, organic carbonates, norditerpenic esters, cysteine protease procerain and other chemicals have made this plant a popular subject of study for many years. Although, due to the existence of cardenolides, plants have the potential to be poisonous (cardiac glycosides). Cardenolides were discovered to be most abundant in the latex [17]. According to research, the leaf of this plant consists of cardenolides 162 mg/g at dry weight and 2 mg/g [18].

Recent research indicates that during malarial infections, the concentration of reactive oxygen species (such as superoxide and hydroxyl radicals) rises [19], due to haemoglobin digestion, parasite metabolism and host defense mechanism of the *Pf*-iEs. Reactive oxygen species plays a crucial role in various physiological processes for both host and parasite, as well as overproduction of intracellular ROS attack and damage lipids, proteins, nucleic acids, and integrity of cell membrane, which affects the survival of the cell and induce, the gradual apoptosis by suppressing the specific gene expressions [20]. To maintain the intracellular redox homeostasis parasite activates the machinery includes enzymes like iron-superoxide synthetase (Fe-SOD), glutathione-S-transferase (GST), glutathione synthetase (GS), γ-glutamylcysteine synthetase (γ-GCS), thioredoxin reductase (TrxR), and peroxiredoxins (nPrx) but, it lacks the catalase and glutathione peroxidase [21]. Therefore, it is understood that oxidative stress has a significant clinical and pathological role in malaria infection [22]. In addition, oxidative stress factor is also an effective therapeutic tool for example quinolines and artemisinin act chiefly via the production of ROS but, resistance development against Quinoline and Artemisinin- Based Combination Therapies (ACTs) in Southeast Asia and various regions of Africa over a long-term usage adds more complications in treating *P. falciparum* infected patients [21].

The goal of the current study is to evaluate the antioxidant activity and potential anti-plasmodial activity of the hydro-alcoholic extract of *L. camara* and *C. procera* leaves as well as to investigate how it affects the stabilisation of the erythrocyte membrane and in reducing the oxidative damage that this parasite onslaught caused in erythrocytes.

#### **2. Methods**

Traditional antimalarial drugs use Reactive Oxygen Species (ROS), which eventually causes their parasiticidal effects. However, the antimalarial activity mediated by ROS is something that *Plasmodium's* evolutionary dynamism actively works to displace [21]. In light of this, current studies have focused on developing natural medicines to reduce *Plasmodium* infections. The inclusion of several secondary phytochemicals endows *L. camara* L. and *C. procera* L. plant extracts with strong therapeutic capabilities.

To perform the phytochemical extraction from the selected plants (*L. camara* and *C. procera),* the following method has been implemented.

#### **2.1 Plant collection and extraction**

Leaves of *L. camara* and *C. procera* were collected from the Gujarat University campus, Ahmedabad, Gujarat. Samples of plant material were authenticated by the Botany Department, Gujarat University, Ahmedabad, India. The plant material was thoroughly cleansed with distilled water, shade dried at ambient temperatures (27–37°C), then manually powdered using a commercial electrical stainless-steel blender. For defattation step, powder was treated with petroleum ether and continuously stirred on a magnetic stirrer for 48 hours. Using a Soxhlet extractor, 20 grammes of defatted powdered plant material were extracted for 72 hours with 200 ml of solvent (hydro-alcoholic: 70:30). At low pressure, the diluted crude solvent that had accumulated in the flask was concentrated. The yield that was obtained after drying was stored at 4°C until use [23].

#### **2.2** *In vitro* **cultivation of chloroquine (CQ )-sensitive strain 3D7 of** *Plasmodium falciparum*

It is obtained from National Institute Malaria Research (NIMR), New Delhi. Erythrocytic stages were developed to determine the correlation between the antiplasmodial efficacy of the plant extracts and the reactive oxygen species generated. Asynchronized *P. falciparum* culture was maintained according to the method described by Trager and Jensen [24] with minor modifications.

#### **2.3 Determination of antioxidant activity of the plant extracts by DPPH assay**

Both the plant extracts were assessed by the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) to determine its antioxidant activity by scavenging the free radicles as per the method reported by Gyamfi *et al*. [25]. EC50 value of the plant extract was evaluated by the dose response curve using Microsoft Excel.

#### **2.4** *In vitro* **study of erythrocyte membrane stabilisation and % inhibition of parasite entry**

In many underdeveloped nations today, medicinal plants represent an important part of the traditional healthcare infrastructure. The bioactive chemicals found in these medicinal plants have been used to create several medications since antiquity [26, 27]. This study also suggests that the extract may alter the erythrocyte membrane in a way that renders red blood cells hostile and incompatible as parasite host cells or that it may prevent the parasite from entering the erythrocyte even when extract is not present in the growing media. *L. camara* and *C. procera* extracts are rich in phenolic and flavonoid components [12, 28].

Erythrocyte membrane stabilisation efficacy of the two plant extracts were assessed using the haemolysis method mentioned by Jansen *et al*. [29] and Linz-Buoy *et al*. [28]. In which, control O+ve erythrocytes were resuspended in 10% PBS (v/v) and incubated with crude plant extracts consisting of the range (1000 μg/ml with serial dilution up to 7.18 μg/ml concentration) under agitation for 24 hours at room temperature, this mixture then subjected to centrifugation for 5 minutes at 10,000 x g followed by the absorbance of the supernatants was measured at 550 nm with a microplate reader.

*Antioxidant Efficacy of Selected Plant Extracts Debilitates the* Plasmodium *Invasion… DOI: http://dx.doi.org/10.5772/intechopen.106844*

#### *2.4.1 In vitro % inhibition of parasite entry into RBCs*

*In vitro* percent inhibition of parasite entry into RBCs was calculated as mentioned in Linz-Buoy *et al*., [12]. For each concentration, growth inhibition was calculated as a percentage of the number of schizonts compared to five untreated controls. Doseresponse curves were used to get the mean IC50 values (percentage of schizonts vs. logarithm of drug concentration).

#### **2.5 Oxidative stress parameters**

By preventing the emergence of new free radical species, stopping radical chain reactions, changing existing free radicals into less dangerous molecules, and repairing oxidative damage, antioxidants work through protective processes at various levels within cells [30].

#### *2.5.1 Cellular sample preparation*

Intracellular ROS: Cells were harvested after 48 hours of incubation at 37°C. Cultured cells were harvested and centrifuged to remove the culture media at 500 g for 10 minutes at 4°C. The pelleted cells were haemolysed in four times volume of ice-cold injection water/1x RBC lysis buffer and centrifuged again at 4°C. This cell lysate was then used to measure intracellular ROS by Metta *et al.* [31] with minor modifications.

#### **2.6 Parameters**

#### *2.6.1 Lipid peroxidation (LPO)*

The method of Okhawa *et al*. [32] was used to measure the quantities of thiobarbutiric acid reactive species (TBARS) in Control erythrocytes, *Pf-*iEs, and Pre-treated Control erythrocytes with the plant extract.

#### *2.6.2 Superoxide dismutase (SOD)*

The NADH-phenazinemethosulphate-nitroblue tetrazolium formazon is the foundation of the superoxide dismutase (SOD) test. The activity of SOD in Control erythrocytes, *Pf-*iEs, and Pre-treated Control erythrocytes with the plant extracts by the method of [33].

#### *2.6.3 Catalase*

As hydrogen peroxide is broken down by the enzyme catalase, its UV absorbance at 240 nm can be used in this method to evaluate enzyme activity. The method of Sinha [34] was employed to determine the activity of catalase enzyme in control erythrocytes, *Pf-*iEs, and Pre-treated Control erythrocytes with the plant extracts. By decrease in absorbance, the activity of an enzyme can be calculated.

#### *2.6.4 Reduced glutathione (GSH)*

The method outlined by Ellaman *et al.* [35] was used to measure the reduced glutathione (GSH) level. When GSH reacts with DTNB (5, 5′-dithiobis nitro benzoic acid), a yellow compound formed. Control erythrocytes, *Pf-*iEs, and Pre-treated Control erythrocytes with the plant extracts were taken as samples.

#### *2.6.5 Glutathione peroxidase (GPx)*

The Rotruck *et al*. [36] method was used to measure the glutathione peroxidase (GPx) enzyme activity in control erythrocytes, *Pf*-iEs, and pre-treated control erythrocytes A cytosolic enzyme called GPx catalyses the conversion of peroxide radicals to alcohols and oxygen as well as the conversion of hydrogen peroxide to water and oxygen.

#### **2.7 %Inhibition of growth proliferation (MTT) assay**

3- (4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) Assay is based on the capacity of Mitochondria succinate dehydrogenase dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) into an insoluble, coloured formazan product which is measured spectrophotometrically at 540 nm [37, 38]. Since reduction of MTT can only occur in metabolically active cells, the level of activity is a measure of the viability/cytotoxicity of the cells.

*In vitro* confirmations of the toxic effect of the phytocomponents have measured on the HeLa cell lines with 6 serial dilutions from 250 to 0.0156 µg/ml. The formazan crystals were formed, following the reduction of MTT by metabolically active (viable) cells.

#### **2.8 Haemoglobin (Hb) determination**

Haemoglobin determination was carried out using a Sahli's hemoglobinometer with standard colour comparator and the final value was recorded as g/dl Haemoglobin content [39]. The amount of Hb of control, *Pf*-iEs and pre-treated erythrocytes is given in **Figure 8**.

#### **3. Results**

#### **3.1 Antioxidant activity of the plant extracts by DPPH assay**

Antioxidant activity demonstrates the plant extract's ability to scavenge Reactive Oxygen Species (ROS) in a dose-dependent manner. The antioxidant potency of two plant extracts is revealed in this study. Ascorbic acid is used as the standard here (**Figure 1**).

As antioxidants are required to protect cells from oxidative stress, the *L. camara and Calotropis Procera* extracts tested in this study were found to be high in antioxidants *L camara* showed the higher antioxidant activity compared to *C. procera* plant extract, with 85.23% at the highest concentration of 250 g/ml. Linz-Buoy *et al*. [28] demonstrated *in vitro* that this effective antioxidant capability is beneficial in this control.

#### **3.2** *In vitro* **erythrocyte membrane stabilisation and % inhibition of parasite entry**

The % erythrocyte membrane stabilisation assay with haemolysis (osmotic fragility test) is shown in **Figure 2**. The property of erythrocyte membrane stabilisation is performed in dose dependent manner i.e., from the highest concentration (250 μg/ml to 7.81 μg/ml).

*Antioxidant Efficacy of Selected Plant Extracts Debilitates the* Plasmodium *Invasion… DOI: http://dx.doi.org/10.5772/intechopen.106844*

#### **Figure 1.**

*Bar graph presentation of antioxidant activity of two selected plant's hydro-alcoholic extracts, compared with the standard ascorbic acid. Values are Mean ± S.E. n = 6. (\* p<0.01, \*\* p < 0.001, \*\*\* p <0.0001).*

#### **Figure 2.**

*The % erythrocyte membrane stabilization with haemolytic assay reported via bar graph presentation of selected hydroalcoholic extracts of the two plants (L. camara and C. procera).*


### **Table 1.**

*% Inhibition of entry of P. falciparum MRC-2 strain in pre-treated erythrocytes with hydro-alcoholic extract of L. camara and C. procera. This shows the hydro-alcoholic extract of L. camara and C. procera were assessed for their anti-plasmodial activity. Here, asynchronized culture of P. falciparum MRC-2 were subjected to pre-treated control erythrocytes for 24 hours.* *Antioxidant Efficacy of Selected Plant Extracts Debilitates the* Plasmodium *Invasion… DOI: http://dx.doi.org/10.5772/intechopen.106844*


### **Table 2.**

*% Inhibition of entry of P. falciparum RKL-9 strain in pre-treated erythrocytes with hydro-alcoholic extract of L. camara and C. procera. This shows the hydro-alcoholic extract of L. camara and C. procera were assessed for their anti-plasmodial activity. Here, asynchronized culture of P. falciparum RKL-9 were subjected to pre-treated control erythrocytes for 24 hours.*

Only 2.48 percent of the erythrocytes were hemolyzed when treated with the hydroalcoholic extract of *L. camara* at the highest concentration (250 g/ml) used in the study. Pretreatment with *C. procera* hydroalcoholic extracts yielded 7.66 percent at the concentration (250 g/ml).

In the pretreatment of control erythrocytes, haemolysis increases as the crude extract concentration decreases.

#### *3.2.1 % Inhibition of parasite entry*

The experimental results of this study showed that pre-treatment of the hydroalcoholic extracts of selected two plants showed anti-plasmodial efficacy for an asynchronized culture of *P. falciparum* MRC2 and RKL-9 (**Tables 1** and **2**).

Among these two plants *L. camara* is found more effective in killing 50% of malaria parasites in the appropriate course of incubation time (24 hours) of the asynchronized parasite of strain MRC-2 and RKL-9 according to the experimental evidence obtained in our study. Among the two plants *L. camara* reported highest antioxidant activity and thus same result reflected in the % erythrocyte membrane stabilisation assays and proves its potent anti-plasmodial activity.

#### **3.3 Oxidative stress parameters**

#### *3.3.1 Lipid peroxidation (LPO)*

When *Pf*-iEs were compared to control erythrocytes, lipid peroxidation was significantly increased (**Figure 3**). The results also revealed that, when compared to the negative control, pre-treated erythrocytes with the plant extracts had significantly lower levels of lipid peroxidation, as evidenced by higher MDA values.

#### **Figure 3.**

*Showing the lipid peroxidation (TBARS) level in control, infected and pre-treated erythrocytes with the hydroalcoholic extracts of L. camara and C. procera. extract. Values are mean ± S.E. # (×104 nmoles of MDA/100mg cells wt/60min).*

*Antioxidant Efficacy of Selected Plant Extracts Debilitates the* Plasmodium *Invasion… DOI: http://dx.doi.org/10.5772/intechopen.106844*

The results also indicated that there was a significant reduction (\*\* p < 0.001) in lipid peroxidation as indicated by the elevated MDA values, for the control erythrocytes when compared to negative control, were treated with both the extracts when subjected to *Pf-MRC2* and *Pf-RKL9* for 24 hours.

#### *3.3.2 Superoxide dismutase (SOD)*

While *Pf*-iEs were compared to control erythrocytes, the activity of superoxide dismutase was found to be significantly lower (p < 0.001). Once control erythrocytes were pre-treated with hydroalcoholic extracts of *L. camara* and *C. procera* and then subjected to *Plasmodium* infection (MRC-2 and RKL-9) for 24 hours, SOD activity increased significantly (**Figure 4**).

#### **Figure 4.**

*SOD enzyme activity in control and Pf-iEs, compared with the pre-treated control erythrocytes with the hydroalcoholic extracts of L. camara and C. procera. The unit of SOD is SOD (units/mg protein). Values are mean ± S.E. \*\* p < 0.001; \* p < 0.01.*

#### *3.3.3 Catalase*

The results obtained show that catalase activity significantly decreases with *Plasmodium* infection but, it is significantly improved with the use of plant extracts (**Figure 5**).

#### *3.3.4 Reduced glutathione (GSH)*

The study revealed a significant (p < 0.01) increase in reduced glutathione level in the *Pf*-iEs. After pre-treatment with the hydroalcoholic extracts of both the plants, decrease in the GSH level was observed (**Figure 6**).

#### **Figure 5.**

*Shows the catalase enzyme activity in control erythrocytes, PfMRC-2 and PfRKL-9 infected and treated control erythrocytes with the hydroalcoholic extracts of L. camara and C. procera. Values are mean ± S.E. Unit of catalase activity measured: mmol of H2O2 consumed/min/mg protein) \*\* p < 0.001; \* p < 0.01.*

#### **Figure 6.**

*Shows the reduced Glutathione content differs during the Plasmodium infection as it increases compared to control erythrocytes but, when pre-treated with hydroalcoholic extracts, decreases the glutathione level. Among two plants L. camara hydroalcoholic extract shows most effective decline. Values are mean ± S.E. \* p < 0.01. The unit of GSH measurement (µg/100mg cells weight).*

#### *3.3.5 Glutathione peroxidase (GPx)*

The study revealed that the *Plasmodium* infection resulted in a considerable decline in GPx activity. Following the application of the plant extracts via *Antioxidant Efficacy of Selected Plant Extracts Debilitates the* Plasmodium *Invasion… DOI: http://dx.doi.org/10.5772/intechopen.106844*

pre-treatment to control erythrocytes, there were noticeable increase seen in the Glutathione peroxidase activity (**Figure 7**).

#### **Figure 7.**

*Showing GPx enzyme activity in control erythrocytes, Pf-iEs and pre-treated erythrocytes with hydroalcoholic extracts of L. camara and C. procera. Values are mean ± S.E. \*\* p < 0.001.*

#### **3.4 % Inhibition of growth proliferation (MTT) assay**

The result decreases of % growth proliferation after pre-treatment with both extracts has tabulated in **Table 3**.


#### **Table 3.**

*Showing the results of MTT assay in which % growth inhibition of extracts of both L. camara and C. procera on the HeLa cells. In vitro confirmation of the toxic effect of the phytocomponents has measured on the HeLa cell lines with 6 dilutions from 250 to 7.81 μg/ml. The formazan crystals were formed, following the reduction of MTT by metabolically active (viable) cells.*

#### **3.5 Haemoglobin (Hb) determination**

The highly significant decrease of Hb content has been observed in the infected erythrocytes (iEs). Compared with the control erythrocytes much significant changes in the amount of Hb has not been observed in the pre-treated erythrocytes (**Figure 8**).

#### **Figure 8.**

*Showing the haemoglobin content in control, Pf-iEs and pre-treated erythrocytes with plant extracts. Values are Mean ± S.E. \*p<0.01 \*\*p<0.001.*

#### **4. Discussion**

Membrane stabilising profiles of various extracts of *L. camara* on bovine red blood cells exposed to both heat and hypotonic induced lysis were reported previously [40]. Earlier studies have shown that various herbal drugs can stabilise the red blood cell membrane [41]. The mode of action of the extracts could relate to binding to the erythrocyte membranes with subsequent alteration of the surface charges of the cells. This might have prevented physical interaction with aggregating agents or promote dispersal by mutual repulsion of like charges which are involved in the haemolysis of red blood cells. It has been reported that certain saponins and flavonoids exerted profound stabilising effect on lysosomal membrane both *in vivo* and *in vitro*, while tannins and saponins possess ability to bind cations, there by stabilising erythrocyte membrane and other biological macro molecules [41]. It is not surprising that *P. falciparum* develops resistance to antimalarial medications whose mode of action is dependent on the production of ROS in a shorter amount of time. This shows a common pathway of resistance to the K13 propeller gene mutation, which is supported by the development of artemisinin resistance to novel endoperoxide-based hybrid molecules [42].

The ROS-managing machinery of the parasite could be disrupted to preserve and improve the actions of the antimalarials, ensuring the ongoing relevance of ROS-producing antimalarials [21]. Moreover, aromatic and therapeutic plants make up much of India's natural resources. According to reports, phenolic and flavonoid compounds function as antioxidants to exert anti-allergic, anti-inflammatory, antidiabetic, antimicrobial, antiviral, antithrombotic, and vasodilatory effects. As a result, they may prevent diseases like cancer, cardio-vascular disease, cataract, eye disorders, and Alzheimer's [43, 44].

Free radicals are vital to many metabolic processes and play a crucial role in aerobic metabolism and life. Reactive oxygen species (ROS) have been implicated in mediating oxidative damage to macromolecules such lipids, proteins, and DNA.

#### *Antioxidant Efficacy of Selected Plant Extracts Debilitates the* Plasmodium *Invasion… DOI: http://dx.doi.org/10.5772/intechopen.106844*

Antioxidants protect cells at multiple levels by inhibiting the formation of free radical species, interfering with radical chain reactions, converting existing free radicals into less harmful molecules, and repairing oxidative damage [30]. Flavonoids and phenolic compounds are abundant in *L. camara* extracts [28]. Flavonoids and tannins are most likely responsible for the free radical scavenging effect. Plant phenolic compounds also serve as primary antioxidants.

Becker *et al.* [45] have shown an increase in the lipid peroxidation of *Plasmodium* infected RBCs. Moreover Erel *et al.* [46] have demonstrated that *plasmodia* succeed in accumulating free radical scavenging enzymes within their own cells but deplete them in red blood cells of the host. Polyphenols (flavonoids) have been known to effectively restrict free radical induced peroxidation of lipid. According to [47] in addition to their protein binding and direct scavenging activity, these potent antioxidants interact with membrane lipids and prevent the access of deleterious molecules across the cell membrane. Thus, the infected erythrocytes, treated with the extracts showed decreased lipid peroxidation, due to the potent antioxidant activity of these extracts.

The current study found a highly significant decrease in the erythrocyte antioxidant machinery, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), indicating the presence of a high amount of ROS. *Plasmodium* erythrocytic stages are subjected to a variety of oxidative stress-inducing events, such as haemoglobin metabolism [48]. *P. falciparum* generates H2O2 within RBCs during haemoglobin degradation [45]. Furthermore, the Fenton reaction exposes intra-erythrocytic parasite stages to increased ROS formation [49, 50]. *P. falciparum*infected erythrocytes produced significantly more hydroxyl (OH) radicals and H2O2 than uninfected erythrocytes, according to Atamna and Ginsburg [51]. *Plasmodium* infection was found to cause a significant increase in lipid peroxidation in red blood cells; however, when treated with a hydro-alcoholic extract of *L. camara*, a significant decrease in LPO in infected RBCs was obtained.

SOD activity in *plasmodium*-infected RBCs was significantly lower than in normal RBCs. Several other studies have found a decrease in SOD activities in erythrocytes in malaria patients [52, 53], which supports the findings. This supports its role as an antioxidant, where levels decreased to counteract oxidative stress. The main characteristics of these changes are changes in erythrocyte GSH content, lipid peroxidation levels, and oxidative stress enzymes like SOD, CAT, and GPx. *L. camara and C. procera* extracts were found to be effective enough to overcome this change and return the cell to normalcy as a result, the *L. camara and C. procera* extracts exhibit enormous potential and promise in controlling *Plasmodium* ingress into the host erythrocytes and further reducing the subsequent oxidative stress.

#### **5. Conclusion**

Herbal products are well thought-out to be symbols of safeguard in comparison to the synthetic product that are regarded as unsafe to human life and environment. Phytochemical and pharmacological studies are conducted on *L. camara* and *C. procera*. Based on the results obtained in the study, it can be inferred that the leaves of *L. camara* are rich sources of lot of secondary metabolites/phytocomponents which can be used as a prophylactic drug against malaria.

Finally, the current investigation's experimental work was focused on using membrane stabilisation to assess the anti-plasmodial activity of *L. camara* hydroalcoholic crude extract. This extract did show evidence of potent phytochemicals with erythrocyte membrane stabilising activity. Furthermore, this study found that *L. camara* has better anti-plasmodial activity than *C. procera* against MRC2 and RKL9 *Plasmodium falciparum* strains, in contrast to synthetic products, which frequently have side effects and are dangerous to human life. This plant extract's potent antioxidant phytochemicals are both effective and relatively safe.

Both extracts of *L. camara* leaves had shown positive results on the effect on erythrocyte membrane stabilisation. They could inhibit the entry of parasites, also show effective antioxidant property, and has no toxic effect due to certain unknown compounds in the crude extracts on the normal cells which is proved by the MTT assay.

### **Acknowledgements**

The data acquisition and analysis: Miss. Urja Joshi. Participate in drafting or revising the work: Dr. Dhara Jani. Contribute to the conception or design of the work:Dr. Linz-Buoy George. Interpretation of data for the work& approve the final version of the work to be published: Dr. Hyacinth Highland.

### **Conflict of interest**

"The authors declare no conflict of interest."

### **Abbreviations**


*Antioxidant Efficacy of Selected Plant Extracts Debilitates the* Plasmodium *Invasion… DOI: http://dx.doi.org/10.5772/intechopen.106844*

#### **Author details**

Urja Joshi1,2\*, Dhara Jani2 , Linz-Bouy George2 and Hyacinth Highland2

1 Department of Biochemistry, School of Sciences, Gujarat University, Ahmedabad, Gujarat, India

2 Department of Zoology, BMTC, and Human Genetics, School of Sciences, Gujarat University, Ahmedabad, Gujarat, India

\*Address all correspondence to: urjajoshi@gujaratuniversity.ac.in

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### *Edited by Pier Paolo Piccaluga*

Malaria remains a leading cause of morbidity and mortality worldwide. Children in Africa and selected regions of Asia and Southern America are especially widely affected. Written by expert research teams, this book describes different aspects of the epidemiology, pathobiology, diagnosis, prevention, and treatment of malaria. Chapters highlight current research as well as the gold standards for diagnosis and treatment of the disease, examining recent advances, remaining pitfalls, and novel ideas about prophylaxis and therapy.

> *Alfonso J. Rodriguez-Morales, Infectious Diseases Series Editor*

Published in London, UK © 2023 IntechOpen © 123dartist / iStock

Malaria - Recent Advances and New Perspectives

IntechOpen Series

Infectious Diseases, Volume 20

Malaria

Recent Advances and New Perspectives

*Edited by Pier Paolo Piccaluga*