CHAPTER 2

REVIEW OF LITERATURE

Section 2.1.

Options and Issues

in

Antimalarial therapies

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Review of Literature

2.1. Options and Issues in Antimalarial therapies

2.1.1 Introduction

More than 40% of the world‘s population, much of it socioeconomically and

politically challenged, live in areas where malaria, alone or together with HIV/AIDS and

tuberculosis, is a significant health risk (1, 2). According to the World Health Organization

(WHO), approximately 250 million clinical cases of malaria occur every year (3). Malaria is

estimated to kill nearly one million people annually, with most of the deaths occurring in

children under 5 years of age in sub-Saharan Africa (4). If children survive multiple

infections, such exposure leads to a natural immunity that limits the severity of the disease

later in life. However, this immunity wanes in the absence of continued exposure to malaria

infections. Additionally, pregnant women and newborns have reduced immunity, and

therefore are vulnerable to severe complications of malaria infection and disease (5, 6).

Malaria is primarily caused by four species of the protozoan parasite Plasmodium: P.

falciparum, P. vivax, P. malariae, and P. ovale, which are transmitted by over 70 species of

Anopheles mosquitoes (7, 8). These parasite species occur sympatrically both in human

populations and within infected individuals, with P. falciparum and P. vivax being the

predominant species (9-11). Approximately 80% of all malaria cases and 90% of malaria-

attributed deaths occur in Africa, and are caused by P. falciparum (3). Outside of Africa, P.

vivax is the most widespread species, occurring largely in Asia, including the Middle East

and the Western Pacific, and in Central and South America (2). This parasite species causes a

relatively less lethal form of the disease compared with P. falciparum (12, 13). Efforts to

eradicate malaria in the 1960s were meet with resistance to anti-malarial drugs and

insecticides and great ecological concerns. The World Health Organization (WHO) has

declared malaria control a global development priority but it is not an easy task for many

reasons. The antimalarial gap required to bring new and affordable drugs is large.

The life cycle of the malaria parasite illustrating (Figure 2.1.1.) the various stages that

are relevant to therapeutic targetting. These are (1) the anopheline mosquito vector, used in

experimental protocols to immunize with irradiated sporozoites administered by mosquito

bite; (2) the sporozoite, the target of several vaccines, including RTS,S; (3) the liver-stage,

usually targeted by vectored vaccines; (4) the blood-stage, usually targeted by protein in

adjuvant vaccine candidates. Merozoite antigens have been most often included in blood-

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stage vaccines; (5) the gametocyte which along with the ookinete, formed after fertilization in

the mosquito midgut, is the source of parasite antigens used in sexual-stage transmission-

blocking vaccines. Pre-erythrocytic vaccines, which target the sporozoite and the liver-stage

parasite are intended to prevent infection as well as disease while blood-stage vaccines are

intended to prevent clinical illness and death.

Fig 2.1.1.: Life cycle of the malaria parasite [Adapted from 14]

2.1.2 Treatment of Malaria

Effective quinine monotherapy for malaria has been available for more than 350

years, (15) well before the causative organisms of the disease were identified (16). After

World War II, the golden age for antimalarial monotherapy was signalled by widespread use

of fairly cheap and highly effective aminoquinolines, both as prophylactic and therapeutic

agents (17). However, only 15 years after they were adopted, aminoquinoline monotherapies

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for Plasmodium falciparum malaria began to stop working simultaneously in southeast Asia

and in Brazil, (18) and resistance spread rapidly. Perhaps surprisingly, quinine has retained

much of its efficacy, although it can no longer be used as monotherapy for uncomplicated

malaria in Southeast Asia (19). The development of folate antagonists, both as prophylactic

and therapeutic agents, allowed only a short respite before true multidrug resistant malaria

emerged.

Multidrug resistant parasites are the biggest therapeutic challenge to health care in

most malaria-endemic areas. Resistance to chloroquine, the former first-line treatment, is also

becoming apparent in P vivax (20-22). The consequences of antimalarial drug resistance are

stark (23). When the cheapest affordable antimalarial does not work, an alternative regimen

might simply be too expensive. When treatments do not cure patients, more serious morbidity

results, even in initially uncomplicated cases of malaria.

New antimalarial regimens, urgently needed because of the worldwide development

of multidrug resistance, should aim to cure patients no longer responding to standard

therapies. Such regimens should be incorporated into strategies aimed at controlling the

spread of disease, such as insecticide spraying, use of impregnated bed nets, sustained

chemoprophylaxis, and intermittent prophylactic treatment. Since none of these other

measures is used routinely in most parts of Africa and because a vaccine is unlikely to

emerge within the next decade, early and effective chemotherapy for malaria has a pivotal

role in reducing morbidity and mortality.

2.1.3. Antimalarial therapeutic agents

Today when many mosquito vectors are resistant to insecticides (24, 25) and an

effective vaccine is not yet available (24, 26) chemoprophylaxis/chemotherapy remains the

principal means of combating malaria. Over the past 60 to 70 years, since the introduction of

synthetic antimalarials, only a small number of compounds, belonging to three broad classes,

have been found suitable for clinical usage (27, 28). These classes are described below.

2.1.3.1 Quinine and related drugs

Quinine, originally extracted from cinchona bark in the early 1800s, along with its

dextroisomer quinidine, is still one of the most important drugs for the treatment of

uncomplicated malaria, and often the drug of last resort for the treatment of severe malaria.

Chloroquine (CQ), a 4-aminoquinoline derivative of quinine, has been the most successful,

inexpensive, and therefore the most widely used antimalarial drug since the 1940s. However,

its usefulness has rapidly declined in those parts of the world where CQ-resistant strains of P.

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falciparum and P. vivax have emerged and are now widespread. Amodiaquine, an analogue

of CQ, is a pro-drug that relies on its active metabolite monodesethyl-amodiaquine, and is

still effective in areas of Africa, but not in regions of South America. Other quinine-related,

commonly used drugs include mefloquine, a 4-quinoline-methanol derivative of quinine, and

the 8-aminoquinoline derivative, primaquine; the latter is specifically used for eliminating

relapse causing, latent hepatic forms (hypnozoites) of P. vivax. Halofantrine and

lumefantrine, both structurally related to quinine, were found to be effective against multi-

drug-resistant falciparum malaria (27, 29, 30). While lumefantrine, in combination with an

artemisinin derivative, artemether (Coartem), is recommended by the WHO for treating

uncomplicated falciparum malaria, halofantrine generally is not recommended because of

serious side effects and extensive cross-resistance with mefloquine (31).

2.1.3.2 Antifolate combination drugs

Antifolate drugs include various combinations of dihydrofolate reductase enzyme

(DHFR) inhibitors, such as pyrimethamine, proguanil, and chlorproguanil, and

dihydropteroate synthase enzyme (DHPS) inhibitors, such as sulfadoxine and dapsone. With

rapidly growing sulfadoxine-pyrimethamine (SP) resistance, a new combination drug,

Lapdap (chlorproguanil-dapsone), was tested in Africa in the early 2000s, but was withdrawn

in 2008 because of hemolytic anemia in patients with glucose-6-phosphate dehydrogenase

enzyme (G6PD) deficiency (32).

2.1.3.3 Artemisinin and its analogues

Artemisinin drugs, which originated from the Chinese herb qing hao (Artemisia

annua), belong to a unique class of compounds, the sesquiterpene lactone endoperoxides (28).

The parent compound of this class is artemisinin (quinghaosu), whereas dihydroartemisinin

(DHA), artesunate, artemether, and β-arteether are the most common derivatives of

artemisinin; DHA is the main bioactive metabolite of all artemisinin derivatives (artesunate,

artemether, β-arteether, etc.), and is also available as a drug itself (27, 28, 30).

Since 2001, the WHO has recommended the use of artemisinin-based combination

therapies (ACTs) for treating falciparum malaria in all countries where resistance to

monotherapies or non-artemisinin combination therapies (e.g., SP) is prevalent (33). The

rationale for the use of ACTs is based on the facts that artemisinin derivatives are highly

potent and fast acting, and that the partner drug in ACT has a long half-life, which allows

killing the parasites that may have escaped the artemisinin inhibition. Thus, it is thought that

ACTs will delay the onset of resistance by acting as a ―double-edged sword‖ (28, 34, 35).

ACTs, such as artemether- lumefantrine, artesunate-amodiaquine, and artesunate-mefloquine

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are being used in China, Southeast Asia, many parts of Africa, and some parts of South

America (27, 28, 30). Introduction of ACTs has initiated noticeable reduction in malaria

prevalence in these endemic regions of the world (28). Although the mechanism(s) of action

is poorly understood (described below), the current high level of interest in artemisinin drugs

is due to their well-recognized pharmacological advantages: These drugs act rapidly upon

asexual blood stages of CQ-sensitive as well as CQ-resistant strains of both P. falciparum and

P. vivax, and reduce the parasite biomass very quickly, by about 4-logs for each asexual

cycle. In addition, these drugs are gametocytocidal. Thus, through rapid killing of asexual

blood stages and developing gametocytes, artemisinin drugs significantly limit the

transmission potential of the treated infections. These drugs have large therapeutic windows,

and based on extensive human use, they appear to be safe, even in children and mid/late

pregnant women. Furthermore, there is no reported ―added toxicity‖ when these drugs are

used in combination with other types of antimalarial compounds.

In addition to these three main classes of compounds, the antibiotic tetracycline and

its derivatives, such as doxycycline, are consistently active against all species of malaria, and

in combination with quinine, are particularly useful for the treatment of severe falciparum

malaria (27). Until recently, a combination of atovaquone, a hydroxynaphthoquinone, and

proguanil (Malarone) was considered to be effective against CQ- and multidrug-resistant

falciparum malaria; atovaquone resistance has recently been noticed in Africa (36).

Piperaquine, another member of the 4-aminoquinoline group, in combination with DHA

(Artekin), holds the promise of being successful in CQ-resistant endemic areas of Southeast

Asia (27, 28, 30).

2.1.3.4 Antimalarial Drug Resistance

This limited antimalarial armament is now severely compromised because of the

parasite‘s remarkable ability to develop resistance to these compounds (39, 37). In many

different malaria-endemic areas, low to high-level resistance in the predominant malaria

parasites, P. falciparum and P. vivax, has been observed for CQ, amodiaquine, mefloquine,

primaquine, and SP. Plasmodium falciparum has developed resistance to nearly all

antimalarial drugs in current use, although the geographic distribution of resistance to any

one particular drug varies greatly. In particular, Southeast Asia has a highly variable

distribution of falciparum drug resistance; some areas have a high prevalence of complete

resistance to multiple drugs, while elsewhere there is a spectrum of sensitivity to various

drugs (39). Until 2009, no noticeable clinical resistance to artemisinin drugs was reported.

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However, as described below, a number of recent studies have raised concerns about the

efficacy of ACTs, particularly in Southeast Asia.

2.1.3.5 Genetic Basis of Drug Resistance for Antimalarials

It is believed that the selection of parasites harboring polymorphisms, particularly

point mutations, associated with reduced drug sensitivity, is the primary basis for drug

resistance in malaria parasites (38, 39). Drug-resistant parasites are more likely to be selected

if parasite populations are exposed to sub-therapeutic drug concentrations through (a)

unregulated drug use; (b) the use of inadequate drug regimens; and/or (c) the use of long half-

life drugs singly or in non-artemisinin combination therapies. In recent years, significant

progress has been made to understand the genetic/molecular mechanisms underlying drug

resistance in malaria parasites (40, 41). Chloroquine resistance (CQR) in P. falciparum is now

linked to point mutations in the chloroquine resistance transporter (PfCRT [encoded by pfcrt,

located on chromosome 7]). Pfcrt-K76T mutation confers resistance in-vitro, and is the most

reliable molecular marker for CQR. Polymorphisms, including copy number variation and

point mutations, in another parasite transporter, multidrug resistance (PfMDR1 or Pgh1

[encoded by pfmdr1, located on chromosome 5]), contribute to the parasite‘s susceptibility to

a variety of antimalarial drugs. Point mutations in pfmdr1 play a modulatory role in CQR,

which appears to be a parasite strain-dependent phenomenon (42).

Point mutations in the P. falciparum DHPS enzyme (encoded by pf-dhps, located on

chromosome 8) are involved in the mechanism of resistance to the sulfa class of

antimalarials, and accumulation of mutations in the P. falciparum DHFR domain (encoded by

pf-dhfr, located on chromosome 4) defines the major mechanism of high-level

pyrimethamine resistance. In field studies, a pf-dhps double mutant (437G with either 540E

or 581G), combined with the pf-dhfr triple mutant, was found to be frequently associated

with SP treatment failure (28, 29). Orthologues of pfcrt (pvcrt-o), pfmdr1 (pvmdr1), pf-dhps

(pv-dhps), and pf-dhfr (pv-dhfr) in P. vivax have been identified and found to be

polymorphic. However, associations of the mutant alleles of pvcrt-o/pvmdr1 and pv-dhps/pv-

dhfr with clinical resistance to CQ and SP, respectively, are unclear (43).

2.1.4 Emergence of Artemisinin Resistance in P. falciparum

A major advance in the search for effective treatment for drug-resistant malaria came

with the discovery of artemisinin and its derivatives. These compounds show very rapid

parasite clearance times and faster fever resolution than any other currently licensed

antimalarial drug (28). Given the recent significant impact of ACTs on malaria morbidity and

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mortality, it is worrisome that higher recrudescence rates of P. falciparum malaria after

artemisinin treatment are seen in some areas. Recrudescence, the reappearance of an infection

after a period of quiescence, occurs in up to 30% of patients on artemisinin monotherapy, and

in up to 10% of patients on ACTs (18, 34). The underlying mechanism of recrudescence after

artemisinin treatment is unclear. As illustrated by recent in-vitro studies, the occurrence of

parasite dormancy, where parasites enter a temporary growth-arrested state, may provide a

plausible explanation for this phenomenon (45, 46).

Furthermore, there are recent concerns that the efficacy of ACTs has declined near the

Thai-Cambodia border, a historical ―hot spot‖ for emergence and evolution of multidrug-

resistant malaria parasites (47). In this region, artemisinin resistance is characterized by

significantly slower parasite clearance in-vivo, with or without corresponding reductions on

conventional in-vitro susceptibility testing (48, 49). A recent heritability study found that the

observed artemisinin-resistant phenotype of the parasites has a genetic basis (50). The study

showed that genetically identical parasite strains, defined by microsatellite typing, strongly

clustered in patients with slow versus faster parasite clearance rates. It was suggested that the

artemisinin-resistant phenotype is expected to spread within parasite populations that live

where artemisinins are deployed unless associated fitness costs of the putative resistance

mutation(s) outweigh selective benefits. The genetic basis for artemisinin resistance is not

known at present. The main reason for this lack of knowledge is that the molecular

mechanism(s) of action of artemisinin drugs remains uncertain and debatable, although a

number of potential targets have been proposed (51, 52). Investigations into potential protein

targets of artemisinin drugs have included PfATP6, a P. falciparum SERCA-type calcium-

dependent ATPase in the endoplasmic reticulum (43); PfCRT; PfMDR1; P. falciparum

multidrug resistance-associated protein 1 (PfMRP1), residing on the parasite plasma

membrane; a putative deubiquitinating protease termed UBP-1; and mitochondrial proteins

(51, 52). A recent study, conducted on artemisinin-resistant P. falciparum strains from

western Cambodia, did not find any correlation between artemisinin-resistant phenotype and

pfmdr1 mutations/amplification, as well as mutations in pfatp6 (or pfserca), pfcrt, ubp-1, or

mtDNA (54).

At present there is no concrete evidence of artemisinin resistance occurring elsewhere.

Preliminary reports of emerging artemisinin resistance in South America and some African

countries are as yet unconfirmed. For now, the situation appears to be confined to the Thai-

Cambodia border, and is most likely a result of distinctive features of malaria treatment in

this geographic area. These include unregulated artemisinin monotherapy for more than 30

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years, creating massive drug pressure, and counterfeited or substandard tablets that contain

less active ingredients than stated (55). However, if the problem is not urgently and

appropriately addressed, it is feared that artemisinin resistance could spread worldwide.

Given the pivotal role that artemisinin drugs now play, this would be a major blow for

malaria treatment and control, since there are currently no alternative drugs to replace them.

Even the development pipeline of new drugs consists predominantly of ACTs.

2.1.5 Global Pipeline of Antimalarial Drugs

Olliaro and Wells (56) reviewed the global pipeline of new antimalarial combinations

and chemical entities, in various stages of development till February 2009. Overall, the

pipeline consists of 22 projects, either completed (artemether-lumefantrine and artesunate-

amodiaquine), in various clinical stages (phase III/registration stage, n = 4; phase II, n = 8;

phase I, n = 5), or in preclinical translational stage (n = 3). Chemical novelty is relatively low

among the products in clinical stages of development; the prevailing families are of

artemisinin-type and quinoline compounds, mostly in combination with each other. Among

the non-artemisinin and/or non-quinoline combinations and single compounds are:

Azithromycin-CQ (phase III), fosmidomycin-clindamycin (phase II), methylene blue-

amodiaquine (phase II), methylene blue-chloroquine SAR 97276 (a choline uptake

antagonist, phase II), and tinidazole (an anti-parasitic nitroimidazole compound, phase II).

It is clear from the size of the antimalarial drug development pipeline that the pipeline

has not reached critical mass, which is of concern particularly when we consider the recent

emergence of artemisinin resistance, and the apparent decrease in time to resistance to each

new drug/drug combination. The challenges for now and for the future are: (i) how to ensure

that we have compounds to combat emerging and future antimalarial drug resistance; and (ii)

how to initiate and expedite the development of compounds that are as innovative as possible

with respect to their chemical scaffold and molecular target.

Section 2.2.

Antimalarial Combination Therapies

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2.2 Antimalarial Combination Therapies: Histrorical background, current

status and future trends

The world health organization has endorsed Artemisinin based Combination

Therapies (ACTs) as first line treatment in malaria. The available marketed antimalarial

combinations combine the rapid schizontocidal activity of an artemisinin derivative

(artesunate, artemether or dihydroartemisinin) with a longer half-life partner drugs viz.

mefloquine, lumefantrine, halofantrine and piperaquine. The major problems associated with

currently available semisynthetic artemisinin derivatives are low oral bioavailability, time

dependent pharmacokinetics (major cause of recrudescence), toxicity and high cost (due to

poor yield of the artemisinin extraction process) etc.

2.2.1 Why combine antimalarials?

The simplest reason for combining antimalarials is to increase efficacy. Additionally,

drug combinations can shorten duration of treatment, hence increasing compliance, and

decrease the risk of resistant parasites arising through mutation during therapy.

The notion that combination regimens might reduce build-up of resistance is based on

work done with mycobacterial infection. In brief, if there is an underlying mutation rate that

confers resistance to antimalarial A at a pre-existing frequency of, for example, one in 108

parasites and to drug B at a frequency of, for instance, one in 1012 parasites, then the chances

of a parasite containing resistance genes to both drugs is one in 1020, providing unlinked

mendelian inheritance applies. Thus, millions (108) of infections are needed before a parasite

resistant to both drugs is encountered, because there are at most 1012 parasites in an adult

with uncomplicated malaria, and even fewer in children. In practice, however, there are many

reasons why this theory might be limited (57), and why both monotherapy and combination

regimens are not as effective as they should be.

2.2.2 Why do combination regimens not always work?

2.2.2.1 Complex genome of parasite

P falciparum has a genome of 2.3×107 bp, (58) compared with Mycobacterium

tuberculosis, which has a genome of only 4.4×106 bp (59). Furthermore, the eukaryotic

falciparum genome contains more complex mechanisms that might mediate drug resistance

than does the bacterium. For example, P falciparum multidrug resistance gene 1 (pfmdr1) is

implicated in resistance to diverse antimalarials, such as the aminoquinolines chloroquine and

amodiaquine and mefloquine and halofantrine (60-61).

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pfmdr1 encodes the protein P glycoprotein homologue (Pgh), which belongs to the

ATP binding cassette family of transporters. These proteins use energy derived from ATP to

transport various structurally unrelated substrates out of the cell. Pgh localises to the

parasite‘s food vacuole membrane where another protein associated with chloroquine

resistance, P falciparum chloroquine resistance transporter (pfcrt), is also found. These

transport proteins probably interact to alter parasite susceptibility to antimalarials (62). Thus,

variations in pfmdr1 are associated with clinically important multidrug resistance phenotypes,

even if the exact mechanisms are not yet understood.

The concept of unlinked resistance genes obeying mendelian rules cannot apply to a

mechanism that depends on changes in pfmdr1 copy number and determines resistance to

structurally unrelated antimalarials.

2.2.2.2 Speed of acquisition of drug resistance

Mutation rates vary dependent on the area of the parasite‘s genome targeted by a

drug. Hotspots for recombination events can change predictions with respect to the speed at

which drug resistance manifests itself. For example, the antimalarial atovaquone targets the

cytochrome bc1 mitochondrial complex, which is subject to a high frequency of pre-existing

mutations. In one study, (63) one in three patients treated with atovaquone monotherapy in

Thailand already carried parasites resistant to the drug. Furthermore, in-vitro, the rate at

which resistance to unrelated antimalarials develops varies considerably, with multidrug-

resistant parasites often developing resistance to unrelated compounds at rates far higher than

those noted for parasites still sensitive to most drugs (64).

2.2.2.3 Undefined mechanisms of survival

Nakazawa and colleagues (65) showed that parasites can survive in culture for up to

10 days despite addition of antimalarial drugs to the medium. Furthermore, the parasites

showed no genetic evidence for resistance after exposure to pyrimethamine or mefloquine,

suggesting some other mechanism of resistance.

Factors such as stage specificity and rate of antimalarial action might affect efficacy

of drugs. For example, artemisinins take several hours to exert maximum antimalarial effects,

particularly on very early stages of parasite development, but have fairly short elimination

half-times (66). Conventional once-daily dosing regimens might therefore allow a small,

undetectable proportion of parasites to escape, even if artemisinins are used for 7 days, (67)

when theory suggests parasites should have been cleared. In this instance, survival of the

parasite does not depend on development of resistance to a drug, and therapeutic outcomes

might be improved greatly by simply altering dose and duration of treatment.

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Also, the number of parasites exposed to a drug is an important risk factor for

recrudescence, both in-vitro (65) and in-vivo. So care is needed when extrapolating results

from in-vitro assays, which involve about 106 parasites per well, to patients, in whom many

more parasites accumulate.

2.2.2.4 Clearance kinetics and efficacy of antimalarials

Some classes of antimalarials (eg, artemisinins) have more rapid parasite clearance

kinetics than other antimalarials. However, this factor does not necessarily make them a more

effective drug type, since clearance rates do not always faithfully reflect the process of

parasite killing. Although clearance of an individual‘s burden of parasites can easily be

monitored by frequent examination of blood films after treatment, there are limitations to the

conventional parasite clearance estimates—namely, PC50, PC90 (reduction of initial

parasitaemia by 50% or by 90%, respectively), and parasite clearance time (PCT) (66). A

new method, in which the parasite reduction ratio (ratio of parasites after one developmental

cycle or 48 h of antimalarial treatment) is measured may be more reliable (67).

Counting of parasites in peripheral blood films can be misleading for many reasons.

First, the number of parasites in a patient‘s circulation does not always indicate the total

parasite burden. However, this limitation is more of a problem in complicated than in

uncomplicated malaria (66). Second, the mechanism of action of an antimalarial affects how

its efficacy should be assessed in-vivo. Artemisinins, for example, remove ring forms of

parasites and might allow return of pitted erythrocytes to the circulation (68). By contrast,

after quinine treatment, conventional parasite clearance times are consistently longer than

after treatment with artemisinins. Does this mean that artemisinins are better drugs than

quinine? Not necessarily, because a circulating parasite might be alive, injured (fatally), or

dead in these circumstances and its exact state cannot be ascertained by routine microscopy

of fixed samples. Comparisons between quinine and artemisinins might therefore be biased

against the true pharmacodynamic efficacy of quinine.

Some evidence to support such discrepancies between clearance of parasitaemia and

killing of parasites comes from experiments that have measured the viability of parasites ex

vivo, after exposure to different classes of antimalarials (69-70). Furthermore, in children

with cerebral malaria, artemether did not improve mortality or shorten surrogate markers of

disease (such as duration of coma) despite the much quicker disappearance of circulating

parasites than noted with quinine (71). Findings of a meta-analysis of individual patients‘

data drawn from trials that compared artemether and quinine suggested that artemether was

better at preventing death or sequelae, (72) but adequately sized prospective trials are needed

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to confirm this result. Such studies should preferably be undertaken in areas where parasites

are not yet developing resistance to quinine.

Assessment of the effects of a drug like clindamycin could be even harder to interpret,

because clindamycin acts by inhibiting apicoplast function and replication, and its effects

only become visually obvious after the first or second cycle of replication (73-74).

Nevertheless, a much larger proportion of parasites is fatally wounded by this drug after

initial exposure than by those already discussed.

How then should one allow for these differences in mechanisms of action of

antimalarials when using crude parasite clearance indices? Measurement of other

pharmacodynamic variables is potentially important. Alternative methods of assay include

sophisticated tests, such as ex-vivo viability studies, use of other objective markers of host

response to infection (such as fever), or even simple symptom assessments. It is noteworthy

that use of adjunct therapies, such as antipyretics, could affect parasite clearance kinetics,

further confounding interpretation (75-76).

The most important information obtained from watching parasites disappear from the

circulation of a patient with uncomplicated malaria is, therefore, that there is no high-grade

resistance to the antimalarial being used. Assessment of comparative antimalarial efficacy

should, therefore, account for differences in mechanisms of action and also monitor true cure

rates. The cure rates corrected for reinfections by PCR should be ascertained at a minimum of

28 days after treatment with short half-life antimalarials or combinations, and at an even

longer interval if long half-life antimalarials are used, for reasons discussed elsewhere (77).

Endpoints of combination therapies assessed 14 days after treatment, as recommended by

WHO and therefore widely used, are not good enough for assessing efficacy. They might

nevertheless provide useful information about the safety of drugs and higher grades of

resistance.

2.2.3 What constitutes an ideal combination?

The same fundamental requirements for ideal antimalarials apply to those used in

monotherapy as well as in combination regimens. Ideally, a regimen should be safe and well

tolerated, efficacious and effective, orally, rectally, and parenterally applicable, stable,

available as a single dose, effective against all stages of parasite development, not susceptible

to parasite resistance, and cheap. The drugs contained in a regimen should have elimination

half-times that match and no other clinically significant negative pharmacokinetic

interactions (78), have independent modes of action, act synergistically in-vivo, and be

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produced as a fixed-dose combination in a single formulation (panel). When combining

drugs, the potential for harm, caused by bringing together adverse effects of more than one

drug or by generating new risks through unpredictable drug interactions, should be weighed

against perceived benefits.

2.2.3.1 Special patient populations

Treatment of adults, children, pregnant women, uncomplicated malaria, and severe

malaria all depend on individual assessment. These different categories of patient present

individual difficulties in the choice of a single effective and safe antimalarial regimen.

Additionally, pharmacogenomic considerations can affect the use of some antimalarials—eg,

glucose-6-phosphate dehydrogenase deficiency can lead to severe haemolysis in patients

given primaquine or dapsone, and metabolism of proguanil is mainly modulated by

CYP2C19 polymorphisms (78).

In adults and children, unhindered multiplication of parasites can rapidly lead to

hyperparasitaemia, particularly in those in whom factors of innate resistance, such as the

sickle cell trait or the nitric oxide synthase gene polymorphism NOS-2Lambarene, are absent

(79). Genetic and acquired resistance factors also play an important part in determining the

efficacy of antimalarial regimens. For example, adults who live in areas that are

hyperendemic or holoendemic for P falciparum malaria are semi-immune, and can be cured

of malaria with less than optimum treatment regimens (80).

Some drugs, such as tetracyclines, are contraindicated in children and pregnant

women, and cannot therefore be considered for use in combination regimens aimed at this

population. Primiparous women are particularly susceptible to some of the adverse effects of

malaria, such as stillbirth, low birthweight of babies, and maternal anaemia and mortality. For

these patients, chemotherapy poses an urgent problem, since most antimalarials are not

licensed for use in pregnancy.

2.2.3.2. Characteristics of an ideal antimalarial combination

Safe and well tolerated

No serious or fatal adverse events

Well tolerated by most patients to increase likelihood of compliance—combinations that

include quinine, for example, will always be compromised by poor tolerability due to

cinchonism

Efficacious and effective

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Cure rate of more than 90% when assessed at an appropriate follow-up time (≥4 weeks

after start of therapy) in the setting of a clinical trial (often the best circumstance in which

efficacy can by measured)

Effectiveness in practice is almost always less than true efficacy because of many

variables, including poor compliance, inadequate dosing, and lack of funds to purchase a

full treatment course

For public-health initiatives in malaria endemic regions, effectiveness of a regimen

should not fall below 75%

Orally, rectally, and parenterally applicable

Regimen can be used in all circumstances—for outpatients, moderately ill children,

patients who are vomiting, and severe malaria

Stable

No problems with storage at temperatures often encountered in the tropics

Stability of drugs should be confirmed, since this factor is essential in maintaining a high

degree of effectiveness

Single dose

To minimise non-compliance

To allow for home use or administration by untrained healthcare workers

If single dose not possible, simple short regimens (preferably ≤3 days) are most effective

Effective against all stages of parasite development in the human host

Effects on gametocytes might reduce transmission if regimen used extensively

Resistant to parasite resistance

Drugs should act in ways that minimize development or rate of acquisition of resistance

by parasites

Cheap

Maximum cost: €2 per adult and €1 per child

Elimination half-times of drugs should match, and there should be no other clinically

significant negative pharmacokinetic interactions between drugs (78)

Matching elimination kinetics is especially important in areas of high malaria

transmission, because parasites exposed to declining concentrations of one drug in a

combination are more likely to be transmitted if selection for resistance to that drug takes

place

Independent mode of action of drugs

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Reduces chances of resistance

Synergistic action in-vivo

Enhancing efficacy of single drops

Fixed-dose in a single formulation

Avoids risk that only one part of the drug combination is taken

2.2.4 What combinations are available?

2.2.4.1 Artemisinin-containing regimens

Antimalarial combination chemotherapy is widely advocated and WHO, in particular,

encourages the use of artemisinin-containing regimens. In Thailand, when other treatments

were failing, an artesunate+mefloquine combination as first-line treatment was successful and

remains so many years later. In tropical Africa, WHO have pushed for artemether-

lumefantrine and artesunate+amodiaquine combinations. Theoretically, artemisinin-

containing combinations might not only improve cure rates but also reduce the speed at

which resistance develops. Experiments in animals have been used to examine the ability of

particular antimalarial combinations to slow down emergence of resistance, (81-83) but large

clinical studies are few, particularly from areas of high malaria transmission (84). In one

longitudinal study, (85) combination chemotherapies of sulfadoxine-pyrimethamine and

amodiaquine or artesunate that were more effective in initial treatment than sulfadoxine-

pyrimethamine alone, were also more effective when given to treat recrudescences. However,

the frequency of molecular markers of resistance to sulfadoxine-pyrimethamine did not differ

in recrudescent infections after therapy with sulfadoxine-pyrimethamine alone or when used

in combinations that included artesunate (85).

Artemisinin-containing combinations might also decrease transmission of drug-

resistant parasites by reducing the number of gametocytes carried by patients (84, 86-89).

Although an attractive idea, this theory probably applies more in areas of low malaria

transmission—for example, southeast Asia—than in areas where transmission rates are

high— for example, much of Africa. Lending support to this notion are results from a study

by von Seidlein and colleagues, (90) in which artesunate was combined with sulfadoxine-

pyrimethamine; despite use of an artemisinin containing combination, transmission of

falciparum malaria was unaffected.

2.2.4.2 Sulfadoxine-pyrimethamine

Some consider sulfadoxine-pyrimethamine a single drug rather than a combination in

which each component acts independently, since, although pyrimethamine inhibits

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dihydrofolate reductase (DHFR) and sulfadoxine inhibits dihydropteroate synthase (DHPS),

inhibition of both enzymes prevents synthesis of folic acid in parasites. Pyrimethamine is also

structurally related to proguanil, and to cycloguanil and chlorproguanil, other drugs that

inhibit DHFR.

Discovered during World War II, pyrimethamine has good oral bioavailability, is

absorbed in a few hours in children with malaria, and has an elimination half-life of about 80

h. Its use as a prophylactic agent for malaria began half a century ago. However, resistant

parasites emerged within weeks, and half the population carried resistant parasites within 2

years (91). Single point mutations in the DNA of the target enzyme are responsible for

resistance, explaining its rapid emergence (92). Higher grade resistance to pyrimethamine is

associated with accumulation of various mutations at the target site. Parasite DHFR has now

been crystallised (93), permitting more accurate structure-function assessments of analogues

and their target.

When sulfadoxine is used in monotherapy, resistance in parasites also becomes

rapidly apparent and is due to mutations in the gene coding for DHPS (92). Sulfadoxine (or

another sulfa drug acting at the same target) was therefore combined with pyrimethamine to

ensure inhibition of folic acid. Sulfadoxine has good bioavailability and an elimination half-

life of about 120 h in children with malaria.

After chloroquine, sulfadoxine-pyrimethamine was the most commonly used

antimalarial treatment in almost all endemic areas between the 1960s and the 1980s (94-96).

However, because of drug resistance, the combination is now rarely used outside Africa. As

efficacy falters, the risk-benefit ratio for use of sulfadoxine-pyrimethamine is also falling, as

a result of reports of rare occurrences of severe and fatal adverse events—mainly Stevens-

Johnson and Lyell syndromes—as well as even rarer instances of hepatotoxicity and

agranulocytosis almost exclusively described after use in chemoprophylaxis in expatriates or

travellers to the tropics (97). The risk of administering this combination is further raised by

the frequency of serious adverse events, mainly skin reactions, noted in patients infected with

HIV-1 (98). However, sulfadoxine-pyrimethamine continues to be used in many countries in

Africa, predominantly because it is a single-dose treatment and cheaper than the alternatives.

The combination is also under investigation as an intermittent prophylactic treatment

for use in infants (99) and in pregnancy (100). WHO already recommends its use for

intermittent prophylactic treatment in pregnancy in tropical Africa on the basis of results

from a single, placebo-controlled, randomised trial (100). Even in these special patient

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populations, however, the rapidly deteriorating efficacy of sulfadoxine-pyrimethamine

remains a problem.

2.2.4.3 Sulfadoxine-pyrimethamine-chloroquine

Chloroquine is an aminoquinoline, which interferes with the parasite‘s haem

degradative pathway and thereby prevents detoxification of harmful products of metabolism

(101). It was used as the antimalarial treatment of choice for decades in all malaria-endemic

areas and is still used as first-line treatment in Central America and parts of Africa for P

falciparum malaria. Today, P. falciparum is resistant to chloroquine in all endemic areas

except Central America.

The combination of chloroquine with sulfadoxine-pyrimethamine has been

sporadically tried in the past. The results of a review, which summarized the findings from

five trials of sulfadoxine-pyrimethamine + 4-aminoquinolines, showed a quicker resolution of

symptoms with the combination than with sulfadoxine-pyrimethamine alone (102). However,

other data do not encourage the use of this combination on grounds of poor effectiveness

(103-104).

2.2.4.4 Sulfadoxine-pyrimethamine-amodiaquine

Amodiaquine, like chloroquine, leads to annoying pruritus, especially in Africans.

Additionally, it causes agranulocytosis and liver damage in about one in every 2000 white

people taking the drug for chemoprophylaxis (105-106).

This ad-hoc triple combination regimen is used sporadically in some endemic areas,

despite the fact that it combines the potential of rare but fatal adverse events associated with

amodiaquine and with sulfadoxine-pyrimethamine, (97, 107) as observed when the drugs

were used as chemoprophylactic agents in travellers to malaria endemic areas. These

observations have led to the withdrawal of both amodiaquine and sulfadoxine-pyrimethamine

in almost all European countries (108). However, when used to treat malaria, similar serious

(and perhaps allergic) adverse events have not been observed so often, perhaps because such

events only arise after repeated exposure, particularly during chemoprophylaxis. If repeated

exposure is indeed a prerequisite for adverse events, then newer intermittent prophylactic

regimens in infants and pregnant women might carry similar risks. Nevertheless, renewed

interest in this combination for treating African children has been generated by favourable

results from Tanzania64 and elsewhere (109-112).

2.2.4.5 Sulfadoxine-pyrimethamine-quinine

Quinine needs to be administered on 7 consecutive days when given as a

monotherapy, which is not always achievable because of cinchonism (dysphoria, tinnitus,

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nausea) and the very bitter flavour of most salts. However, apart from artemisinins, quinine is

the only drug used to treat severe malaria and, despite widespread use, it (with a combination

partner such as clindamycin or tetracycline in Thailand) retains excellent efficacy.

Quinine-sulfadoxine-pyrimethamine has been used in several trials to improve

efficacy, but with unconvincing results in Brazil (113) and Bangladesh, (114) where parasite

resistance to sulfadoxine-pyrimethamine was already established. In an area where most

parasites are still sensitive to sulfadoxine-pyrimethamine, this drug combined with a 3-day

regimen of quinine might be more effective than either drug alone, as suggested by the results

of a study from South Africa (115).

2.2.4.6 Sulfadoxine-pyrimethamine-mefloquine

This single dose, fixed, triple combination formulation has never been extensively

used in most countries, and was introduced onto the market when the efficacy of sulfadoxine-

pyrimethamine against parasites was already impaired. It is not an additively or

synergistically acting combination, (116) and each drug has a different pharmacokinetic

profile. Mefloquine has a long (about 3 weeks) half-life (78). Other disadvantages are its high

price and moderate safety profile.

2.2.4.7 Atovaquone-proguanil

Atovaquone acts by inhibiting the parasite‘s mitochondrial complex bc1 and

disrupting the membrane potential. Because of the high frequency of resistant mutants

associated with atovaquone monotherapy (116) it was combined with proguanil (a biguanide

acting on DHFR) after promising results of in-vitro experiments, showing synergistic activity

(117). Atovaquone is erratically absorbed since it is fat soluble, and should therefore be taken

with food (not always possible when patients are in the throes of an attack of malaria).

Atovaquone has an elimination half-life of about 73 h in African patients (118). The

elimination half-lives of proguanil and its metabolite cycloguanil are estimated at 14 h (119).

Atovaquone-proguanil has been developed for therapy and prophylaxis of malaria, (120-121)

and shows good safety and tolerability in children and adults, with high efficacy against P.

falciparum malaria when used in a 3-day regimen. However, its use in the tropics is severely

curtailed by its high price.

2.2.4.8 Chlorproguanil-dapsone

Dapsone is a sulfa drug, with an elimination half-life of about 30 h, which has been

combined with chlorproguanil (which has an elimination half-life of about 20 h).

Chlorproguanil-dapsone was developed by a private-public partnership (122) combination

treatment for African children. A phase III multicentre study compared the efficacy and

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safety of chlorproguanil-dapsone with sulfadoxine-pyrimethamine in children in Gabon,

Kenya, Malawi, Nigeria, and Tanzania. (123) Results indicate that the combination is well

tolerated and efficacious. However, there was a higher frequency of serious haematological

adverse events after chlorproguanil-dapsone treatment than after treatment with sulfadoxine-

pyrimethamine (123). The safety of this combination is, therefore, a major concern,

particularly since dapsone can cause methaemoglobinaemia and haemolysis in individuals

with glucose-6-phosphate dehydrogenase deficiency (124). To test for glucose-6-phosphate

dehydrogenase routinely in malaria-endemic areas before giving antimalarials is

impracticable. There are also concerns about the potential for rapid emergence of cross-

resistance to this combination in areas where resistance to sulfadoxine-pyrimethamine is

established or becoming so. In Thailand, for example, efficacy of this combination has been

severely limited for some time (125).

2.2.4.9 Quinine-tetracycline

Tetracycline is an antibiotic that probably acts by inhibiting the binding of aminoacyl

tRNA to the ribosome. It acts fairly slowly (over days rather than hours), is well absorbed

orally, and has an elimination half-time of 8 h, which is similar to that of quinine. Tetracyline

maintains activity against multidrug resistant parasites. In Thailand, a 7-day course of

quinine+-tetracycline, in which the drugs need to be taken three-to-four times a day, has been

used to treat falciparum malaria successfully for decades. A major limiting factor in

compliance has been symptoms of cinchonism, leading to replacement of this combination by

artemisinin-based regimens. Because the use of tetracyclines is contraindicated in children

and pregnant women, use of this combination has always been restricted. Emergence of

parasite resistance over the past 10 years in areas where the combination has been extensively

used has further hampered its use (126-127).

Doxycycline is related to tetracycline but has a longer elimination half-life (about 20

h), allowing once-daily dosing. However, doxycycline, like tetracycline, cannot be used by

children and pregnant women. Although dosing of doxycycline is more convenient than

dosing of tetracycline and therefore compliance might be improved, quinine-doxycycline has

rarely been advocated or used in clinical trials for malaria (128).

2.2.4.10 Quinine-clindamycin

Quinine-clindamycin has never been widely used, although numerous studies show

both good efficacy and sound safety profiles in adults, children, and pregnant women. Results

of studies in South America, (113) Africa, (129) Southeast Asia, (130) and in non-immune

travellers who acquired malaria in different endemic areas, (131) support the efficacy of this

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combination. Both drugs have fairly short plasma half lives (clindamycin about 2–4 h), (132)

perhaps reducing the risk of selecting for resistant parasites. A clear disadvantage is

cinchonism.

2.2.4.11 Artesunate-amodiaquine

Artemisinins are made from extracts of Artemisia annua and rapidly reduce

parasitaemia and relieve symptoms caused by malaria (133). They act by inhibiting a P

falciparum-encoded sarcoplasmic-endoplasmic reticulum calcium ATPase, (53) and not by

inhibiting the haem metabolic pathway as previously supposed (134-135). Most clinically

important artemisinins are metabolised to dihydroartemisinin (elimination half-life of about

45 min), in which form they have comparable antimalarial activity (136). Artemisinins are

generally well tolerated. However, their use in monotherapy is associated with high

incidences of recrudescent infection, (137) suggesting that combination with other

antimalarials might be necessary for maximum efficacy. WHO developed artesunate-

amodiaquine for treatment of malaria in African children through a private–public

partnership. In a multicentre phase III trial in Gabon, Kenya, and Senegal, this new drug

combination showed a better overall efficacy than amodiaquine alone (138). However, 6% of

patients in both groups developed neutropenia, (138) a finding that warrants further

investigations, especially since this adverse event is often associated with amodiaquine (107).

Moreover, as is the case for artesunate-mefloquine and artesunate-sulfadoxine-

pyrimethamine, this combination shows a high degree of pharmacokinetic mismatch. In most

African regions where malaria is hyperendemic, widespread use of this regimen will

necessarily lead to a prolonged exposure of parasites to low doses of amodiaquine and its

active metabolite. Despite the rather disappointing findings of a multicentre trial, (138) WHO

recommends this regimen for treatment of uncomplicated falciparum malaria in African

children. Indeed, in some African countries artesunate+amodiaquine is considered as first-

line treatment for children with uncomplicated malaria.

2.2.4.12 Artesunate-sulfadoxine-pyrimethamine

Artesunate-sulfadoxine-pyrimethamine has also been assessed in African children. It

gave promising results in The Gambia, where it was as well tolerated and as efficacious as

sulfadoxine-pyrimethamine alone (89). However, in further WHO-led trials in African

children, the combination was disappointing (139).

2.2.4.13 Artemether-lumefantrine

Artemether-lumefantrine is the only fixed-dose artemisinin-containing formulation

registered after internationally recognised guidelines. It seemed safe and well tolerated in

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children as well as in adults; (140-141) however, a study showed irreversible hearing

impairment (142). Like atovaquone, lumefantrine absorption is enhanced with food, causing

problems in children with malaria. It is also expensive. Lumefantrine is structurally related to

halofantrine and has a half-life of several days but, unlike halofantrine, does not cause

cardiotoxicity. Like artesunate and mefloquine, there is considerable mismatch in elimination

half-lives of artemether and lumefantrine (143). In early studies, a four dose regimen was not

particularly effective (144-146). These findings led to the recommendation of a more

complex 3-day regimen, which should be given at 0, 8, 24, 36, 48, and 60 h. Efficacy of the

six-dose regimen is adequate, but inferior to the combination of artesunate+mefloquine in

Thailand (147-148).

2.2.4.14 Artesunate-mefloquine

A 3-day regimen of artesunate-mefloquine has been the preferred treatment for

malaria in Thailand for almost a decade (84, 149). It is safe, well tolerated, and highly

effective, (84, 150) and has also been investigated in South America (150) and Africa (151).

In Thailand, malaria incidence and in-vitro mefloquine resistance of parasites has decreased

since this combination has come into use. Disadvantages of this regimen include its price and

the pharmacokinetic mismatch of each drug. In a hyperendemic area, widespread treatment

with this combination would lead to long-term exposure of parasites to low doses of

mefloquine.

2.2.4.15 New advances

Two new combinations have undergone clinical trials. Dihydroartemisinin-

piperaquine is another artemisinincontaining combination (152) that compares favourably in

efficacy (99% cure rate, 164/166 patients) with mefloquine combined with artesunate (99%

cure rate, 76/77 patients) in Vietnamese patients assessed 56 days after treatment (153).

Although dosing may need optimisation, dihydroartemisinin-piperaquine might prove to be

more affordable than other combinations of comparable efficacy.

Fosmidomycin-clindamycin is a new antimalarial combination where both drugs act

on the the parasite‘s apicoplast, and not on targets of conventional antimalarials. In initial

clinical trials, fosmidomycin rapidly eliminated malaria parasites, but did not lead to a

sufficiently high final cure rate when used alone (154-155). However, when fosmidomycin

was combined with the synergistically acting clindamycin, rapid parasite clearance and 100%

cure rates were achieved in Gabonese children at 28 day follow-up (156).

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2.2.5 The future

Multidrug resistance in parasites has forced the use of combination antimalarial

regimens. The need for effective treatments has thrown together antimalarial combinations

that have often worked better than monotherapies, though sometimes only temporarily.

However, we are still far from discovery of an ideal regimen. The best options available now

include: mefloquine-artesunate, which works well in Thailand, although little is known about

how this combination translates to high transmission areas; and clindamycin-quinine, which

is effective in non-immune individuals, suggesting that clindamycin-artesunate should also be

studied. Dihydroartemisinin-piperaquine and fosmidomycin-clindamycin are promising

treatment candidates. Until better regimens become available there are enormous demands on

policy makers to choose between existing combinations, many of which have not been

sufficiently studied for informed judgments to be made.

Section 2.3.

Drug Discovery and Pharmacokinetics

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2.3 Drug Discovery and Pharmacokinetics

Drug research encompasses several diverse disciplines united by a common goal,

namely the development of novel therapeutic agents. The search for new drugs can be

divided functionally into two stages: discovery and development (157). The former consists

of setting up a working hypothesis of the target enzyme of receptor for a particular disease,

establishing suitable models (or surrogate markers) to test biological activities, and screening

the new drug molecules for in-vitro and/or in-vivo biological activities (158). In the

development stage, efforts are focused on evaluation of the toxicity and efficacy of new drug

candidates. Recent surveys indicate that the average new chemical entity taken to market in

the United States requires 10 to 15 years of research and costs more than $300 million (159).

Figure-2.3.1 depicts different phases of drug discovery and development.

The lead identification phase in drug discovery involves identification and

characterization of drug targets, synthesis of new lead molecules together with traditional

chemistry, computer aided drug design and/or combination chemistry and further

characterization of physicochemical characters of promising candidate. The hits are identified

using in-vitro screening assays together with high throughput screening for ‘hit’ generation.

In lead optimization step, chemists use empirical and semi-empirical structure-activity

relationships to modify the chemical structure of a compound to improve the in-vitro activity.

However, good in-vitro activity cannot be extrapolated to good in-vivo activity unless a drug

has good bioavailability and a desirable duration of action. The lead optimization phase

typically include, ADME screening to improve upon the degree of potency, which already

has been achieved (159-160) Further in-vivo screening is performed involving testing of the

candidate drug in suitable animal models, their pharmacokinetic and toxicokinetic profiles.

This whole process from high throughput screening to in-vivo screening constitutes pre-

clinical phase in drug discovery and development. Once the drug reaches this phase, an

investigation IND is filed to the drug regulating authorities. After IND application approval,

the clinical phase starts which involves testing in human subjects, and is divided into 4

phases. Each phase involves process scale-up, pharmacokinetics, drug delivery and drug

safety activities (161).

Phase I

Phase I clinical studies are performed to define initial parameters of toxicity and

tolerance. It also includes study of relevant pharmacokinetics of drug in healthy subjects and

optimization of drug delivery systems. These studies help in finding dose range to help

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establish upper level of tolerability in patient population. Pilot pharmacokinetics,

bioavailability and single dose ADME studies are usually carried out in this phase (162-163).

Phase II

Phase II trials are the first studies in patient population carried out to evaluate the

efficacy in limited patient population and to define both the therapeutic dose range and

dosing regimen for a particular disease. Studies on intra- and inter subject variability, dose

proportionality, multiple dosages ADME are also carried out (162-163).

Phase III

These studies are conducted in considerably large number of patients than phase II

and are frequently carried out on a multi center basis. The principal aims are to demonstrate

long-term safety and tolerance and to compare the new drug with the existing standard

treatment. Possible drug-drug interactions, effects of age, sex and other subject variables on

both pharmacokinetics and pharmacodynamic variables are also studied at this stage. These

studies are conducted while NDA has been filed (162-163).

Phase IV

Phase IV studies are conducted after NDA approval is granted and product license has

been obtained. They are large-scale, long-term trials designed to investigate the incidence of

relatively rare adverse reactions. These studies are also helpful in documenting drug-drug

interactions. Most Phase IV studies are used to extend the range of approved indications for

the drug (162-163).

In quest to reach to the market a new chemical entity (NCE) has to undergo several

stages of screening in preclinical species and human beings for its efficacy and safety, which

has been shown in figure-2.3.1. This process requires approximately $ 800 million and

approximately 10-12 years time which necessitates the decrease in cycle time and cost

effective screening for the maximum benefit of the pharmaceutical industry. Ever decreasing

cycle times and cost cutting provide the impetus for innovative and efficient ways of weeding

out of unwanted compounds from development pipeline (164). About ten years ago, main

reason of failure of the compound was poor pharmacokinetics, but due to advent of high

throughput techniques pharmacokinetic screening could be introduced in the discovery phase,

which drastically reduced the attrition rate due to unfavorable pharmacokinetics. In recent

years, cause of failure has been recognized as preclinical toxicity or lack of therapeutic

efficacy. Metabolism which is defined as chemical transformation of molecules causes an

exposure to unknown entity which might put the subject at potential risk. So, regulatory

bodies have shown growing concern for the impact of drug metabolism and drug-drug

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interaction of drug safety and efficacy (165). Pharmacokinetics and drug metabolism

continue to play an increasingly important role in drug development, starting with drug

discovery and lead optimization, pharmacology and safety evaluation, continuing into clinical

development, and finally helping to position the product in the market place. For this

purpose, the discipline of Pharmacokinetics has become an integral part of modern drug

discovery and development process.

Fig 2.3.1.: Drug discovery and development phases for drug interaction studies

2.3.1 Pharmacokinetics

Pharmacokinetics is the science of the kinetics of drug absorption, distribution, and

elimination (i.e., excretion and metabolism). The description of drug distribution and

elimination is often termed drug disposition. Characterization of drug disposition is an

important prerequisite for determination or modification of dosing regimens for individuals

and groups of patients. The study of pharmacokinetics involves both experimental and

CHEMICAL COMBINATORIAL

A. Discovery

Lead

Identification

High Throughput Screening

Physicochemical Properties

Lead

Optimization

In-vitro Screening

File IND

In-vivo Screening

Phase I

Phase II

Phase III

File NDA

Registration

MARKET

Pre-

Clinical

Phase IV

Clinical

Drug

Interaction

Studies

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theoretical approaches. The experimental aspect of pharmacokinetics involves the

development of biologic sampling techniques, analytical methods for the measurement of

drugs and metabolites, and procedures that facilitate data collection and manipulation. The

theoretical aspect of pharmacokinetics involves the development of pharmacokinetic models

that predict drug disposition after drug administration. Pharmacokinetic process has been

depicted in figure-2.3.2

Absorption is the movement of drug from its site of administration into the systemic

circulation.

Distribution is reversible transfer of a drug between the blood and the extra vascular fluids

and tissues.

Metabolism is the chemical transformation of a drug from one form to another which

normally results in termination of action.

Elimination is the process whereby drugs and/or their metabolites are irreversibly transferred

from internal to external medium (166).

2.3.2 Role of Pharmacokinetics

Pharmacokinetics has become an integral part in drug discovery and development.

Today, pharmacokinetic data makes an important contribution in drug development

Absorption Distribution-Storage Excretion

Release

Release Drug in Solution Urine Bile

Faeces

Sweat

Saliva

Biotransformation Fig 2.3.2.: Pharmacokinetics

Free Drug

Metabolites

Protein Bound

Site of Action

Bound Free

Storage Tissue

Free Bound

Drug in

Dosage

Form

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programme ranging from early discovery to marketing authorization submission to post

marketing surveillance.

Early pharmacokinetic and metabolic evaluation with rapid information feedback is

crucial to obtain optimal pharmacokinetic and pharmacological properties (167). Due to time

constraints and the availability of only small quantities of each compound in the discovery

stage, studies are often limited to one or two animal species. Therefore the selection of

animal species and the experimental design of studies are important in providing a reliable

prediction of drug absorption and elimination in humans.

Pharmacokinetics is the integral of the time coincident process of ADME and is considered as

a powerful screening tool in early drug discovery process.

The events following drug administration can be divided into two phases:

i. Pharmacokinetic phase, in which the adjustable elements of dose, dosage form, frequency

and route of administration are related to drug level-time relationships, and

ii. Pharmacodynamic phase, in which the concentration of drug at the site of action is

related to the magnitude of the effects produced (168).

Magnitude of both the desired actions and toxicity are functions of the drug concentrations at

the site(s) of action.

The ultimate goal of pharmacokinetics is to give patients maximal benefit of the drug

by close and accurate measurement of the drug and/ or its metabolites in different biological

matrices, thus offering optimal drug management and patient care (169).

The use of pharmacokinetics for better patient care includes:

Individualization of patient dose and dosing regimen.

Assessment of the bioavailability and bioequivalence of the drug by the proposed

routes.

Aid in determining the mechanisms of drug-drug interactions and their avoidance.

Prediction of pharmacokinetics in man, from results obtained in animals.

Identification of optimum methods to accelerate drug elimination from the body in the

case toxicity and/ or over dosage.

Identification of active metabolites of drugs and quantification of their role in

producing the overall response following drug administration.

2.3.3 Key pharmacokinetic parameters: Theory and rationale

2.3.3.1 Bioavailability

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The bioavailability of a drug is defined as the fraction of the ingested or administered

dose that is available to the systemic circulation. Thus, both absorption and elimination

process rules the bioavailability of a given drug..

As a general rule, oral bioavailability can be estimated as:

F = Fa Fg Fh Fi

where F refers to systemic oral bioavailability, Fa is the fraction absorbed across intestinal

wall and Fg Fh Fi is the product of fractions escaping clearance by the GIT, liver and lung.

The product of the fraction available after gut and liver extraction (Fg Fh) after oral

administration primarily determines the oral clearance of a drug, though the contribution of

lung clearance need to be evaluated.

2.3.3.2 Clearance

The best estimate for drug elimination can be obtained by determining total clearance

of the drug. Organ clearance is defined as the volume of blood that must be cleared of a drug

in unit time in order to account for the rate of drug elimination. Thus, clearance is the ratio of

elimination rate of the drug to the drug concentration in the blood entering the organ. The

total clearance is calculated as the sum of all individual organ clearances of the drug.

Assuming liver and /or kidney as major organs involved in drug elimination prediction of

hepatic and renal clearances are considered most important in predicting pharmacokinetic

characteristics of new chemical entities.

2.3.3.3 Volume of Distribution

The volume of distribution is a measure of the extent of drug distribution and is

determined by the binding of the drug in plasma as well as in tissues. Since it is assumed that

only the unbound drug can diffuse across membranes, it is implicit that distribution to tissues

is affected by plasma protein binding. It is also important to understand that because of

significant tissue binding for most drugs, the apparent volume of distribution far exceeds the

total body water. As such, the volume of distribution is a proportionality constant relating the

drug concentration in blood or plasma to the amount of drug in the body.

Vd = Vb fb + Ve ft (Anderson et al.) (63)

where Vd is the volume of distribution, Vb is the blood volume, Ve is the extra vascular tissue

space volume, fb is the unbound fraction in blood and ft is the unbound fraction in tissues.

2.3.3.4 Elimination Half Life

The half-life (t1/2) of a drug is related to its apparent volume of distribution (Vd) and

systemic clearance (Cl) as per the following equation:

t1/2 = 0.693 (Vd / Cl).

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Thus, the half-life of any drug is a function of its blood and tissue binding as well as its total

clearance and is a derived parameter from Cl and Vd. For drugs with high clearance, the half-

life is relatively independent of changes in intrinsic clearance, whereas for drugs with low

clearance, increase in intrinsic clearance results in decreased half-lives.

2.3.4 Pharmacokinetic screening of NCEs

2.3.4.1 Absorption

Both the rate and extent to which the therapeutic moiety is absorbed is important.

Whatever the route of administration is, a precise pharmacokinetic analysis of the plasma

profile provides data on Bioavailability, Cmax, Tmax and other pharmacokinetic parameters

(170).

2.3.4.2 Distribution

The percentage and characteristics of binding to serum proteins is studied using

appropriate in-vitro methods. Factors which might alter protein binding and so alter

therapeutic response are studied for substances highly bound to plasma proteins. Since only

unbound drug is pharmacologically active; therefore the assessment of bound fraction by the

estimation of plasma protein binding of a compound is another important parameter to be

explored in-vitro (170).

2.3.4.3 Metabolism

Extensive metabolism is generally considered a liability as it limits the systemic

exposure and shortens the half life of a compound. The pharmacokinetic data on such

metabolites, the rate of their formation, elimination, distribution and clearance characteristics

should be known. It is equally important to identify the metabolic pathways of the drug

candidates by conducting in-vitro CYP450 reaction phenotyping assays. The identification of

drug metabolizing enzymes involved in the major metabolic pathways of a compound helps

in predicting the probable drug-drug interactions. In-vitro CYP450 inhibition and induction

screens are used to evaluate the potential of compound towards drug-drug interactions (170).

2.3.4.4 Excretion

The urinary excretion can be defined by parameters such as: Total cumulated amounts

of unchanged and metabolized substance found in the urine following a single dose, also

known as renal clearance of the substance.

The overall result of the pharmacokinetic screening is a substantial increase in the rate

of target identification, generation of new leads, and finally optimization of those leads into

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clinical candidates. ADME studies have always played a critical role in helping to optimize

the pharmacokinetic properties of new drugs thereby increasing their success rate (170).

Section 2.4.

Selected Drugs for Potential Antimalarial Combination Therapy

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2.4. Selected Drugs for Potential Antimalarial Combination Therapy

2.4.1. Lumefantrine

Lumefantrine is a racemic fluorine derivative and originally called Benflumetol. It

was synthesized in the 1970s by the Academy of Military Medical Sciences, Beijing and

registered in China for use as an antimalarial drug in 1987 (171). Lumefantrine is

commercially available in combination with artemether as Coartem®/Riamet® for the

treatment of Plasmodium falciparum malaria. Coartem® is well-tolerated, fast-acting with

96% cure rate in the treatment of uncomplicated falciparum malaria (172-176). Artemisinin

based combination therapy (ACT) is regarded as a first line treatment to overcome the

problem of emerging resistance. ACTs make combined use of the rapid schizontocidal

activity of an artemisinin derivative with a longer half-life partner drug. Lumefantrine is an

antimalarial drug with the long duration of action and high cure rate and has proved to be

among the best partner drug in artemisinin based combination therapy such as coartem®

(176-178).

Absorption of lumefantrine, a highly lipophilic compound, starts after a lag-time of up

to two hours, with peak plasma concentration about 6-8 hours after dosing. The absorption of

lumefantrine is highly influenced by lipids and food intake (from 10% by fasten to 100% at

normal diet). Lumefantrine is eliminated very slowly with a terminal half-life of 2-3 days in

healthy volunteers and 4-6 days in patients with falciparum malaria (140, 143, 175, 179). Its

plasma protein binding is almost 100% (180). Lumefantrine is N-debutylated, mainly by

CYP3A4, in human liver microsomes to desbutyl-lumefantrine (DLF). The in-vitro

antiparasitic effect of desbutyl-lumefantrine is 5 to 8 fold higher than lumefantrine (172).

2.4.1.1. Physicochemical Properties

Lumefantrine, 2-(dibutylamino)-1-[(9E)-2,7-dichloro-9-[(4-chlorophenyl)

methylidene] fluoren-4-yl] ethanol (Figure 2.4.1.) is an arylamino alcohol (140). Its

molecular weight is 528.939 g/mol. The compound is an odorless yellow powder. It is poorly

soluble in water, oils and most organic solvents but is soluble in unsaturated fatty acids (171)

and acidified methanol. It is a lipophillic compound with low intrinsic clearance and erratic

oral variability and therapeutic levels are more reliably achieved by co-administration with a

fatty meal (140,143,181-184).

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2.4.1.2. Bioanalytical Methods for Quantitative Analysis of Lumefantrine in Plasma

In total, there are ten published methods for quantitative analysis of lumefantrine in

iomatrices using liquid chromatography (LC) with ultraviolet (UV) or tandem mass

spectrometric (MS/MS) detection (Table 1). The first publications describing quantitative

analysis of lumefantrine were published in year 1996 in the international literature (178). It

uses liquid-liquid extraction (LLE) in combination with liquid chromatography and UV

detection. Acetic acid and water were added to methanol in order to optimize the retention

behavior but at the same time peak shape was poor and broad. Further, the peak shape was

improved remarkably by addition of small amount of diethylamine in mobile phase (178).

Mansor et al. (177) described a method using liquid-liquid extraction. Retention

behavior and peak shape was optimized using mobile phase containing 10% acetonitrile in

ammonium acetate buffer (pH 4.9, 0.1M) on a C18 reversed-phase Partisil 10 ODS-3

Phenomenex column.

After nine years of first two methods utilizing LLE, a bioanalytical quantitative

analysis of lumefantrine and its desbutyl-metabolite utilizing HPLC and solid-phase

extraction (SPE) on octylsilica column was presented by Lindegardh et al. (176)

Chromatographic separation of lumefantrine and its desbutyl-metabolite is carried out on a

SB-CN Agilent column and acceptable peak symmetry can be achieved with a simple

acetonitrile-phosphate buffer mobile phase. The addition of sodium perchlorate to the mobile

phase was found to be very efficient for altering the separation between the analytes and

Fig. 2.4.1.: Lumefantrine

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interfering endogenous compounds. The perchlorate-ion forms an ion pair with the charged

analytes and thereby increases their retention while the neutral endogenous compounds

remain unaffected.

In the same year, Annberg et al. (173) also reported bioanalytical method employing

LC-UV detection and SPE sample preparation technique on octylsilica column, using the

same mobile phase and column as reported by Lindegardh et al. The merit of the method

reported by Annberg et al. over the method reported by Lindegardh et al. is use of 96-

wellplate format for determination of lumefantrine with shorter turn around time enabling

high throughput analysis.

Blessborn et al. (174) reported automated SPE and liquid chromatographic method for

the determination of lumefantrine in capillary blood on sampling paper as its among the most

feasible and applicable way to collect and store samples from patients in clinical field studies.

Whatman 31 ET Chrsampling paper was pre-treated with 0.75 M tartaric acid before

sampling capillary blood to enable a high recovery of lumefantrine. The sampling paper was

treated with glacial acetic acid and acetonitrile, then further processed using SPE and finally

quantified with HPLC on Zorbax SB-CN column. The method used similar LC and SPE

settings, with some modifications, as in the previous developed method by Lindegardh et al.

for Lumefantrine.

In the year 2008, Ntale et al. (185) described HPLC quantification method for

lumefantrine and its desbutyl metabolite in whole blood spotted on filter paper utilizing LLE

with di-isopropylether along with small amounts of methanol, phosphate buffer and

potassium hydroxide. Good peak symmetry is achieved with a mobile phase containing 10%

acetonitrile in ammonium acetate buffer on Zorbax Eclipse XDB-Phenyl column.

In 2010, Huang et al. (186) reported HPLC-UV method for lumefantrine

quantification in human plasma utilizing protein precipitation followed by solid phase

extraction sample preparation method. Fine tuning of the sample preparation method

improved the recovery of LF significantly. Gradient elution followed by a wash phase with a

high percentage of organic solvent minimized interference from carryover impurities. TFA

(0.1%), instead of phosphate salt, added to the mobile phase minimized peak tailing.

Recently, Hodel et al. (187) developed an interesting liquid chromatography–tandem

mass spectrometry (LC–MS/MS) method for the simultaneous determination of 14

antimalarial drugs and their metabolites which are the components of the current first-line

combination treatments for malaria (artemether, artesunate, dihydroartemisinin, amodiaquine,

N-desethyl-amodiaquine, lumefantrine, desbutyllumefantrine, piperaquine, pyronaridine,

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mefloquine, chloroquine, quinine, pyrimethamine and sulfadoxine). Extraction was done by a

combination of protein precipitation, evaporation and reconstitution in methanol:ammonium

formate 20mM (pH 4.0):: 1:1. Reverse-phase chromatographic separation of antimalarial

drugs is obtained using a gradient elution of 20 mM ammonium formate and acetonitrile both

containing 0.5% formic acid, followed by rinsing and reequilibration to the initial solvent

composition up to 21 min. Separations were done on a Atlantis® C18 Waters column to

provide good electro-spray sensitivity and acceptable peak symmetry. The chromatographic

system was coupled to a triple stage quadrupole Quantum Ion Max mass spectrometer.

Analyte quantification is performed by electro-spray ionization–triple quadrupole mass

spectrometry by selected reaction monitoring detection in the positive mode. The retention

time reported for lumefantrine and desbutyllumefantrine is 15 minutes and 13 minutes,

respectively.

2.4.1.3. Discussion

The method reported by Zeng et al. on HPLC-UV platform, has reasonable sensitivity

for many therapeutic monitoring applications. However, Liquid chromatography coupled to

atmospheric pressure ionization tandem mass spectrometry is currently the method of choice

for the quantitative determination of drugs in biological matrices. The advantages of this

technique include high specificity, sensitivity and throughput resulting in increased

popula