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ONAKU, LINDA O.
PG/ M.PHARM/09/51916
PG/M. Sc/09/51723
EVALUATION OF COMBINATIONS OF ARTESUNIC ACID
AND AQUEOUS EXTRACT OF Azadirachta indica OR Carica
papaya ON THE REDUCTION OF PARASITEMIA IN MICE
INFECTED WITH Plasmodium berghei
PHARMACEUTICAL SCIENCES
A THESIS SUBMITTED TO THE DEPARTMENT OF PHARMCEUTICS, FACULTY OF
PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA
Webmaster
Digitally Signed by Webmaster’s Name
DN : CN = Webmaster’s name O= University of Nigeria, Nsukka
OU = Innovation Centre
NOVEMBER, 2010
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EVALUATION OF COMBINATIONS OF ARTESUNIC ACID AND AQUEOUS
EXTRACT OF Azadirachta indica OR Carica papaya ON THE REDUCTION OF
PARASITEMIA IN MICE INFECTED WITH Plasmodium berghei
BY
ONAKU, LINDA O.
PG/ M.PHARM/09/51916
A PROJECT REPORT SUBMITTED TO THE DEPARTMENT OF
PHARMACEUTICS, FACULTY OF PHARMACEUTICAL SCIENCES, IN
PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF
MASTER OF PHARMACY (M.PHARM) DEGREE OF THE UNIVERSITY
OF NIGERIA NSUKKA.
NOVEMBER, 2010
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CERTIFICATION
This is to certify that Onaku Linda Onyeka, a postgraduate student in the
Department of Pharmaceutics, University of Nigeria Nsukka, with registration
number, PG/ M.PHARM/09/51916, has satisfactorily completed the research work for
the award of the Master of Pharmacy degree in pharmaceutics. The work embodied in
this project report is original and has not been submitted in part or full for any
diploma or degree in this or any other university.
Prof. A. A. Attama Date Prof. V. C. Okore Date
(SUPERVISOR) (CO-SUPERVISOR)
………………………………………………
Prof. A. A. Attama Date
(HEAD OF DEPARTMENT)
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DEDICATION
This research project is dedicated, first, to God; and then my father, Arch. P. C.
Onaku and my Mum, Lady Ify Onaku.
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ACKNOWLEDGEMENT
The Lord has been my light and my help, and I am forever grateful to Him for making
this work see the light of the day.
The contributions of my lecturers, siblings, fellow students and friends to my
academic growth have been considerable. But, the guidance, sacrifice, patience, as
well as moral and academic support of my supervisors, Prof. A. A. Attama and Prof.
V. C. Okore, is deeply appreciated. The contributions of Prof. C. O. Esimone,
Department of Pharmaceutical Microbiology and Biotechnology, Nnamdi Azikiwe
University, who spearheaded this project, are deeply appreciated.
I am forever indebted to my parents, Arch. P. C. Onaku and Lady Ify Onaku and my
uncle, Dr. Peter Oforah, whose moral and financial support has seen me through life
and especially through this project.
As far as this project is concerned, I would like to thank Dr. A. Y. Tijani, NIPRD,
Abuja, and Mr. Ngene, Faculty of Veterinary Medicine, UNN, whose assistance in
obtaining the Plasmodium berghei and determining the parasitemia levels
respectively, shall be bountifully rewarded by God.
Special thanks go to the entire staff of the Department of Pharmaceutics, UNN,
especially Mr. Ogboso Kalu (Expert), Mr. Gugu Thaddeus and Mr. Muogbo Chijioke,
and my mentor and friend, Prof. S. I. Ofoefule, for their assistance and advice.
Onaku, Linda .O
November, 2010.
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TABLE OF CONTENT
Title page - - - - - - - - - i
Certification - - - - - - - - - ii
Dedication - - - - - - - - - iii
Acknowledgement - - - - - - - - iv
Table of content- - - - - - - - - v
Abstract - - - - - - - - - ix
CHAPTER ONE: INTRODUCTION
1.1 Malaria - - - - - - - 1
1.1.1 Life cycle of malaria - - - - - - 4
1.1.2 Pathophysiology of malaria - - - - - 10
1.1.3 Biochemistry of the malaria parasite - - - - 11
1.1.3.1 Carbohydrate metabolism - - - - - 11
1.1.3.2 Lipid metabolism - - - - - - 12
1.1.3.3 Protein metabolism - - - - - - 12
1.1.3.4 Nucleotides and nucleic acid metabolism - - - 13
1.1.3.5 Vitamins and co-factors metabolism - - - - 13
1.1.3.6 Heme metabolism - - - - - - 14
1.1.4 The malaria parasite genome - - - - - 14
1.1.5 Diagnosis of malaria - - - - - - 15
1.2 Prevention of malaria - - - - - - 18
1.3 Treatment of malaria - - - - - 23
1.3.1 Drugs used in the treatment of malaria - - - 24
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1.3.2 Artesunate - - - - - - 27
1.3.2.1 Mechanism of action of artesunate - - - - 29
1.3.2.2 Pharmacokinetics of artesunate - - - - 29
1.3.2.3 Pharmacological actions of artesunate - - - 29
1.3.2.4 Side effects of artesunate - - - - 30
1.3.2.5 Artemisinin-based combination therapy - - - 30
1.4 Pharmacodynamic interaction - - - - 31
1.5 Antimalarial drug resistance - - - - - 33
1.6 WHO guildelines for treatment of uncomplicated malaria - 38
1.7 Plants with antimalarial activity - - - - 43
1.7.1 Antimalarial activity of Carica papaya - - - 45
1.7.2 Antimalarial activity of Azadirachta indica - - - 46
1.8 Methods employed in the evaluation of antimalarial
activity of a substance - - - - - - 49
1.8.1 In vitro methods for screening antimalarial compounds - 49
1.8.2 In vivo methods for screening antimalarial compounds - 50
1.8.2.1 Plasmodium berghei 4 day suppression test - - - 51
1.9 Objective of the study- - - - - - 52
CHAPTER TWO: MATERIALS AND METHODS
2.1 Materials- - - - - - - - 54
2.1.1 Experimental animals - - - - - - 54
2.1.2 The parasites - - - - - - - 54
2.1.3 Plants extract - - - - - - - 54
2.1.4 Chemicals- - - - - - - - 55
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2.2 Methods - - - - - - - - 55
2.2.1 Chemical preparation - - - - - - 55
2.2.1.1 Extraction of crude drugs - - - - - 55
2.2.1.2 Preparation of artesunate - - - - - 58
2.2.2 In vivo schizontocidal activity of combination of a fixed
dose of artesunate with varying doses of crude extracts - 58
2.2.3 Determination of ED50 of the crude extracts - - - 59
2.2.4 Determination of the kind of pharmacodynamic interaction
between the pure drug and plant extract - - - 60
2.2.5 Data analysis - - - - - - 60
CHAPTER THREE: RESULTS AND DISCUSSION
3.1 Chemical Preparation - - - - - - 61
3.1.1 Percentage yield of crude drugs - - - - 61
3.1.2 Preparation of stock solution of artesunate - - - 61
3.2 In vivo schizontocidal activity of combination of a fixed
dose of artesunate with varying doses of crude extracts - 62
3.2 Survival time and percentage cure of P. berghei infected
mice after treatment - - - - - - 70
3.4 Determination of ED50 of the crude extracts - - - 74
3.5 Determination of the kind of pharmacodynamic interaction
between the pure drug and plant extracts- - - - 78
CHAPTER FOUR: CONCLUSION - - - - - 82
References - - - - - - - - 84
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Appendices - - - - - - - - - 106
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ABSTRACT
Fresh leaves of Azadirachta indica, and mature fresh leaves of Carica papaya were
separately homogenized in sterile cold distilled water for 24 hrs. During the study
each extract was stored in the fridge for maximum of seven days. The extraction was
done prior to the determination of the in vivo schizontocidal activity of the crude
extracts (NCE and PCE), and artesunic acid, and fresh extracts were made as needed.
The Peter’s 4-day suppressive test was the model used in this study. The ED50 and
ED90 were calculated from the dose-response relationship and the values obtained
were used to categorize the antimalarial activity of the crude extracts and the
Pharmacodynamic interaction of the combinations of the crude extracts and artesunic
acid. The average percentage yield of NCE (8.33 %) was higher than that of PCE
(5.42 %). It was found that a one –dose combination of 1000 mg/kg of NCE and 15
mg/kg of artesunic acid, produced a significant reduction of parasitemia (96.87 %)
when compared to artesunic acid alone at a dose of 20mg/kg (68.14 %). The
combination of 50 mg/kg of PCE and 15 mg/kg of artesunic acid produced a
significant reduction of parasitemia (81.25 %) when compared to 50 mg/kg of PCE
alone (37.70%). The ED50 of NCE and PCE showed that they have moderate and very
good activity respectively. The isobolar equivalent (IE) calculated from the ED90 of
NCE and PCE in combination with artesunic acid showed that the interaction between
artesunic acid and NCE is synergistic, while the interaction between artesunic acid
and PCE is antagonistic. The combinations of NCE and PCE with artesunate
produced a cure, while the given dose of artesunate did not produce cure during the
30-day period of the study. These results have showed that antiplasmodial
combinations of an artemisinin derivative and aqueous extract of neem leaf (NCE) are
possible, providing a potentiated reduction of parasitemia, and increased cure rate.
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CHAPTER ONE
INTRODUCTION
1.1 Malaria
Malaria is an acute and chronic mosquito-borne disease of man caused by a
eukaryotic protist of the genus Plasmodium. The disease is characterized by chills and
fever, anemia, splenomegaly and damage to other organs such as the liver and brain
(1). Malaria is one of the world’s leading killers. Half of the world’s population –
about 3.3 billion people- is at risk. About 1.2 billion of these are considered to be in
high risk areas: Africa or South East Asia. Malaria has a greater morbidity and
mortality record than any other infectious disease (2, 3, 4). The incidence of mortality
is greatest in children and pregnant women (4). Reports have shown that in 2006, 86
% of the 247 million cases of malaria occurred in Africa. Over half of these cases
occurred in Nigeria, Democratic Republic of Congo, Ethiopia, United Republic of
Tanzania and Kenya. Out of the 881000 deaths that occurred from the disease, 91 %
were in Africa, and 85 % of these were children below 5 years (2). The disease has
negative impact on the economy of malaria- endemic countries (5, 6). Indeed HIV
also has a negative impact on the economy of a nation. HIV infection and malaria
complement each other. HIV infection increases a person’s susceptibility to malaria
infection, while malaria increases viral load of HIV patients (7). According to Centers
for Disease Control and Prevention, in 2008, an estimated 190 - 311 million cases of
malaria occurred worldwide and 708,000 - 1,003,000 people died, most of them
young children in sub-Saharan Africa (8).
Five species of the plasmodium parasite can infect humans and they are transmitted
naturally by the bite of an infected female anopheles mosquito. The most serious
forms of the disease are caused by Plasmodium falciparum. Malaria caused by
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Plasmodium vivax, Plasmodium ovale and Plasmodium malariae causes milder
disease in humans that is not generally fatal. A fifth species, Plasmodium knowlesi, is
a zoonosis that causes malaria in macaques but can also infect humans (9, 10).
Typically, one species will cause malaria, but mixed infections do occur.
The life-cycle of plasmodia involves a sexual phase in the female mosquito and an
asexual stage in man. The asexual stage in man includes; the liver stage (tissue
schizonts), erythrocytic stage (erythrocytic schizonts); where the erythrocytes will
burst at intervals and bring about the febrile attack of malaria, and the gametocytic
stage, where the gametocytes (male and female gametes) also develop in some
infected erythrocytes, thus, this stage is responsible for the continual transmission of
malaria, as when the female mosquito feeds, they take in the erythrocyte housed
gametes, which then initiate the sexual stage. Therefore, a compound acting on the
gametes is very important.
Symptoms of malaria include; fever, shivering, arthralgia (joint pain), vomiting,
anemia (caused by hemolysis), hemoglobinuria, retinal damage, (11) and convulsions.
The classic symptom of malaria is cyclical occurrence of sudden coldness followed by
rigor and then fever and sweating lasting three to eight hours, occurring every two
days in P. vivax and P. ovale infections, while every three days for P. malariae (1).
P. falciparum can have recurrent fever every 36–48 hours or a less pronounced and
almost continuous fever. For reasons that are poorly understood, but that may be
related to high intracranial pressure, children with malaria frequently exhibit
abnormal posturing, a sign indicating severe brain damage (12). Malaria has been
found to cause cognitive impairments, especially in children. It causes widespread
anemia during a period of rapid brain development and also direct brain damage. This
neurologic damage results from cerebral malaria and children are more vulnerable
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(13, 14). Cerebral malaria, a complication of severe malaria is associated with retinal
whitening, (15) which may be a useful clinical sign in distinguishing malaria from
other causes of fever (11).
Severe malaria is almost exclusively caused by P. falciparum infection, and usually
arises 6–14 days after infection. Consequences of severe malaria include coma and
death if untreated—young children and pregnant women are especially vulnerable.
Splenomegaly (enlarged spleen), severe headache, cerebral ischemia, hepatomegaly
(enlarged liver), hypoglycemia, and hemoglobinuria with renal failure may occur.
Renal failure is a feature of black water fever, where hemoglobin from lysed red
blood cells leaks into the urine. Severe malaria can progress extremely rapidly and
cause death within hours or days (16). In the most severe cases of the disease, fatality
rates can exceed 20%, even with intensive care and treatment (17). In endemic areas,
treatment is often less satisfactory and the overall fatality rate for all cases of malaria
can be as high as one in ten (18). Over the longer term, developmental impairments
have been documented in children who have suffered episodes of severe malaria (19).
Chronic malaria is seen in both P. vivax and P. ovale, but not in P. falciparum
infections. Here, the disease can relapse months or years after exposure, due to the
presence of latent parasites in the liver. Describing a case of malaria as cured by
observing the disappearance of parasites from the bloodstream can, therefore, be
deceptive. The longest incubation period reported for a P. vivax infection is 30 years
(16). Approximately one in five of P. vivax malaria cases in temperate areas involve
overwintering by hypnozoites (i.e., relapses begin the year after the mosquito bite)
(20).
Pregnant women are especially attractive to the mosquitoes, (21) and malaria in
pregnant women is an important cause of stillbirths, infant mortality and low birth
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weight, (22) particularly in P. falciparum infection, but also in other species infection,
such as P. vivax (23). Normal immune responses are reduced during pregnancy. In
areas of stable malaria transmission like Nigeria, a pregnant woman would have
acquired a partial immunity enough to protect against serious clinical falciparum
malaria, but heavy parasitic infection of the placenta and often severe anaemia do
occur leading to low birth weights of the baby, who may not even survive. First
pregnancies are at a greater risk. In areas of unstable malaria transmission, pregnant
women have no protective immunity and are at serious risk of developing severe life-
threatening falciparum malaria, especially in the last few months of pregnancy and for
several weeks after delivery. Untreated infection in this case can result in abortion,
still-birth, premature labour or low birth weight. Cerebral malaria, pulmonary oedema
and hypoglycaemia frequently occur. This situation is similar in all pregnancies in this
region (24).
1.1.1 Life cycle of the malaria parasite
Malaria parasites are members of the genus Plasmodium (Phylum Apicomplexa). In
humans, malaria is caused by P. falciparum, P. malariae, P. ovale, P. vivax and P.
knowlesi. (10, 25) P. falciparum is the most common cause of infection and is
responsible for about 80% of all malaria cases and about 90% of the deaths from
malaria (26). Parasitic Plasmodium species also infect birds, reptiles, monkeys,
chimpanzees and rodents (27). There have been documented human infections with
several simian species of malaria, namely P. knowlesi, P. inui, P. cynomolgi, (28) P.
simiovale, P. brazilianum, P. schwetzi and P. simium. However, with the exception of
P. knowlesi, these are mostly of limited public health importance (29). P. knowlesi
resembles P. malariae morphologically, but unlike P. malariae its parasite numbers in
the blood is high and clinical symptoms is more severe (24).
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Malaria in humans develops via two phases: an exoerythrocytic and an erythrocytic
phase. The exoerythrocytic phase involves infection of the hepatic system, or liver,
whereas the erythrocytic phase involves infection of the erythrocytes, or red blood
cells. The exoerythrocytic phase does not produce any symptom: It is the erythrocytic
phase that produces symptoms. Malaria parasites are introduced as sporozoites,
present in the saliva of the infected mosquito, into the human body via the bite of the
female anopheles mosquito as they attempt to suck blood needed for them to lay eggs.
About 68 species of the 400 Anopheles spp. can transmit malaria, but the most
efficient vectors belong to the A. gambiae complex, which is widely distributed in
tropical Africa. The mosquitoes are attracted by increased CO2 concentration, warmth
of skin, moisture (breath), lactic acid, urine, feaces and most importantly, a number of
substances called kairomones. The normal body flora produces these kairomones.
But, malaria transmission can also occur congenitally or via inoculation of infected
blood (e.g. blood transfusion).
Within 30 min the sporozoites pass into the liver, their entry into the liver cell being
facilitated by circumsporozoite protein (CSP). The sporozoite carry out exo-
erythrocytic reproduction, i.e. an asexual reproduction called schizogony. Here, the
parasite grows and undergoes several nuclear divisions without the cytoplasm
dividing until it reaches a diameter of 30-70 nm, and this stage or form of parasite is
called liver schizonts. Division of the cytoplasm of the multicellular liver schizonts
occurs giving rise to thousands of merozoites (offspring) (24).
The infected liver cell burst after sometime to release merozoites into the blood
stream. The minimal time required from infection to the appearance of the first
merozoite, which lasts for 6-15 days, is called the prepatent period (30). Incubation
period is the minimal time from infection to appearance of signs and symptoms of
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malaria, and is somewhat longer than the prepatent period. In the case of P.vivax and
P. ovale, the prepatent period extend to typically 6-12 months, as some liver cells do
not burst and will contain parasites called hypnozoites for a long time and they cause
new attacks of the disease if reactivated (recrudescence). They are also responsible for
delayed exacerbations of the disease post-treatment.
The released merozoites bind to the surface of the erythrocytes via merozoite surface
protein -1 (MSP-1) present on their membranes and then penetrate the red blood cell
and remain in a vacuole there. The MSP-1 is highly variable and the parasite may
change structural features within the course of a single infection. This enables it to
carry out immune evasion. The life-cycle is depicted in the diagram below.
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Figure 1: Lifecycle of Plasmodium spp.- Ref 31
Merozoites undergo differentiation into trophozoites (ring form) that then feed on
heamoglobin. The haemozoin formed can be seen after 12-24 h as malaria pigment.
The vacuole of the trophozoites disappears as the parasite becomes older.
Trophozoites reproduce asexually to form schizont (multinucleated parasite).
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Schizonts divide to form merozoites (about 8-24 per schizont) within 48 h (as for P.
vivax, P. ovale and P. falciparium) or 72 h (as for P. malariae). Infected red blood
cells burst after a while to release merozoites which penetrates new erythrocytes in
seconds. The bursting is accompanied by a bout of fever, but the fever will not usually
follow a typical pattern as in every 48 or 72 h as all parasites cannot be at the same
stage of development.
Some merozoites transform into male and female gametocytes after a few days.
Gametocytes do not cause symptoms but are responsible for transmission of the
disease. Female anopheles mosquito may ingest gametocytes as they suck, and the
gametes once ingested divide mitotically three times and develop several flagellae
(exflagellation). Microgametes are formed after 10 min, from one male gametocyte. A
fall in temperature and gametocyte activating factor (xanthurenic acid, present at a
higher concentration in mosquitoes than in the human blood) triggers exflagellation.
The female gametocyte undergoes a slight change to produce macrogametes.
Microgamete and macrogametes will fuse to form a diploid zygote (parasite is
haploid). This zygote then undergoes meiosis to give four haploid parasites, which
later become motile and are called ookinete. The ookinete penetrates a membrane to
pass from the mosquito’s midgut to the intestinal wall, and it migrates through this
wall to the outside of the intestine. Here, the ookinete changes into an immobile form
called oocyst. After a week of repeated mitotic nuclear division in the oocyst,
countless of fusiform parasite called sporozoites are produced, which after rupture of
the oocyst will migrate to the mosquito’s thoracic three-lobed salivary glands.
Sporozoites mature in the salivary gland and are ready to be injected into a human
during a blood meal (24, 32).
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The parasite is relatively protected from attack by the body's immune system because
for most of its human life cycle it resides within the liver and blood cells and is
relatively invisible to immune surveillance. However, circulating infected blood cells
are destroyed in the spleen. To avoid this fate, the P. falciparum parasite displays
adhesive proteins on the surface of the infected blood cells, causing the blood cells to
stick to the walls of small blood vessels, thereby sequestering the parasite from
passage through the general circulation and the spleen (33). This "stickiness" is the
main factor giving rise to hemorrhagic complications of malaria. High endothelial
venules (the smallest branches of the circulatory system) can be blocked by the
attachment of masses of these infected red blood cells. The blockage of these vessels
causes symptoms such as in placental and cerebral malaria. In cerebral malaria the
sequestrated red blood cells can breach the blood brain barrier possibly leading to
coma (34).
Although the red blood cell surface adhesive proteins called Plasmodium falciparum
erythrocyte membrane protein 1 (PfEMP1) are exposed to the immune system, they
do not serve as good immune targets, because of their extreme diversity; there are at
least 60 variations of the protein within a single parasite and effectively limitless
versions within parasite populations (33). The parasite switches between a broad
repertoire of PfEMP1 surface proteins, thus staying one step ahead of the pursuing
immune system.
1.1.2 Pathophysiology of malaria
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When the schizonts in the erythrocytes mature and rupture they release merozoites,
cellular debris and pigments which stimulates the secretion of cytokines from
leucocytes and other cells. This then causes the fever characteristic of malaria attack.
The fever is usually severe and irregular or continuous at the early stage of infection,
but subsequent attack may be milder and regular, every 72 h for P. malariae, or every
48 h for P. ovale and P.vivax. Typical fever attack starts with a cold stage (rigor)
where the patient shivers and feels cold, although his or her temperature is rising. This
is followed by a hot stage where the patient’s temperature rises to maximum and he or
she feels severe headache, arthralgia, back pains, and the patient may have diarrhea
and may vomit. The last stage of this fever attack is called the wet stage, whence the
patient perspires, the temperature falls, pains and headache are relieved and the
patient feels exhausted. There is always some degree of splenomegaly and anaemia,
and jaundice may occur but are particularly seen in falciparum malaria. Other
pathological changes occur depending on the species of plasmodium causing the
malaria.
Malaria caused by P. falciparum was formerly called malignant tertian malaria. The
untreated infection causes fever, splenomegaly, gastrointestinal disturbances, edema,
anaemia, postural hypotension, mental confusion, profound weakness etc.
Cytoadherence of parasitized red blood cells do occur, where these red blood cells
adhere to one another and to the walls of the capillaries of the brain, muscle, kidney,
placenta etc., leading to congestion, hypoxia, and blockage and rupturing of small
blood vessels. If left untreated, severe malaria results, where the parasite attains high
level of parasitemia (up to 30-40 %); leading to hepatomegaly, jaundice, renal failure,
cerebral malaria, black water fever, delirium, coma and death. P. falciparum has no
latent liver form, so that relapse is due to recrudescence.
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Malaria caused by P. vivax and P. ovale are called vivax and ovale malaria
respectively. Their infections are rarely life threatening and parasitemia levels rarely
exceed 2 %. Relapse is due to latent liver forms.
Malaria caused by P. malariae is called malariae malaria or quartan malaria. Its
parasitemia level is usually below 1 %, but its exoerythrocytic stage can last as long
as 30-40 days, and a serious complication called nephrotic syndrome do occur mostly
in children. This syndrome is caused by the deposition of antigen-antibody complexes
on the glomerular basement membrane of the kidney. It progresses to renal failure and
produces edema, marked proteinuria and low serum albumin level (1, 24).
1.1.3 Biochemistry of the malaria parasite
Malaria parasite obtains nutrients from its environment for the production of other
molecules and energy (catabolism), these are then used to maintain the homeostasis of
the parasite and for anabolism (growth and reproduction). Their metabolic pathways
differ from, but are intertwined with that of the human host, because of the intimate
relationship between the host and the parasite. These pathways and the enzymes
involved can be exploited in the design of therapeutic agent, viz; many antimalarials
affect the food vacuole of the parasite, a special organelle for the digestion of host
hemoglobin (35, 36). Its metabolic pathways are summarized below.
1.1.3.1 Carbohydrate metabolism
The blood stage of the parasite actively ferments glucose as a primary source of
energy through glycolysis. The process of glycolysis is similar to that of other
organism, with lactate being the end product. The infected erythrocyte utilizes 75
times more glucose than uninfected erythrocytes. Patients with severe malaria will
thus suffer hypoglycemia, and coupled with their lack of appetite will lead to
convulsion and shock. Enzymes of the pentose phosphate pathway have also been
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identified and provide some of the ribose sugars needed for nucleotide metabolism
and regeneration of reduced NADPH, utilized for biosynthesis or defense against
reactive oxygen intermediates (ROI).
Pyruvate from the glycolytic pathway is employed in an incomplete TCA cycle by the
blood stages of the parasite and a complete TCA cycle by the mosquito-borne stage.
The Hydrogen atoms produced here are employed in oxidative phosphorylation and
the CO2 produced is a by-product. Atovaquone has been shown to inhibit electron
transport and to collapse the mitochondrial membrane potential (37, 38).
1.1.3.2 Lipid metabolism
Malaria parasite needs a huge demand of lipid to grow and its lipid metabolism can be
targeted. The parasite has enzymes that are associated with type II fatty acid synthetic
pathway (also found in plants and prokaryotes) that appear to be located in the
apicoplast. The apicoplast is involved in biosynthesis of fatty acids, isoprenoid
precursors and heme. They also have enzymes involved in lipid synthesis from
glycerides and fatty acids, and in the remodeling of lipid polar head (39).
1.1.3.3 Protein metabolism
The blood stage obtains amino acids for protein synthesis from 3 sources;
I. Degradation of ingested haemoglobin (most abundant source of amino acids).
Up to 60 -80 % of the host total haemoglobin is digested into amino acids. At
20 % parasitemia, 110 g of haemoglobin will be consumed during 48 hrs.
II. De novo synthesis. The parasite can fix CO2 and so synthesize alanine,
aspartate and glutamate.
III. Uptake of free amino acid from the host plasma (or cells) (36, 40).
1.1.3.4 Nucleotides and nucleic acid metabolism
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Malaria parasites obtain preformed purines by salvage pathways and synthesize
pyrimidines de novo. The primary purine salvaged is hypoxanthine from the host
plasma. For de novo synthesis of pyrimidine, bicarbonates and glutamine are used and
will require co-factors like folates (41).
1.1.3.5 Vitamins and co-factors metabolism
Malaria parasites cannot utilize preformed folate from the host erythrocytes and must
synthesize it from p-aminobenzoic acid, glutamate triphosphate and glutamate. Other
preformed vitamins can be utilized by the parasite. Folic acid and its derivatives are
co-factors in the synthesis of nucleotides and amino acids. A dihydrofolate cycle is
involved in de novo pyrimidine synthesis. Dihydrofolate is reduced to tetrahydrofolate
(this pathway is inhibited by pyrimethamine). The tetrahydrofolate is methylated by
serine hydroxymethyltransferase and the resulting methylene tetrahydrofolate fuctions
as methyl donor. For example, thymidylate synthase catalyses the formation of
deoxythymidylatemonophosphate from deoxyuridinemonophosphate by transferring
the methyl group from methylene tetrahydrofolate and this methylene tetrahydrofolate
is recycled back to dihyrofolate in the process (41).
1.1.3.6 Heme metabolism
Heme, an important component of many enzymes, is synthesized by the parasites de
novo as the heme released from hemoglobin degradation by the parasite cannot be
utilized by it. Free heme is indeed toxic to the parasite as it destabilizes and lyses
parasite membranes, and inhibits the activity of several enzymes. To prevent this, the
heme is detoxified by
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I. Sequestration of the free heme into the hemozoin or the malaria pigment.
II. A degradation reaction facilitated by hydrogen peroxide (an ROI) within the
food vacuole.
III. A glutathione-dependent degradation which occurs in the parasite’s cytoplasm
(42,43, 44).
Chloroquine and other 4-aminoquinolines inhibit hemozoin formation, as well as
other heme degradative processes, thus preventing the detoxification of heme. The
free toxic heme also leads to death of the parasite. Also, the electrons released during
the conversion of iron in the haemoglobin from its ferrous state to its ferric state (in
the heme) promotes the formation of ROIs, which can cause damage to lipids,
proteins and nucleic acids, and thus cellular damage. Superoxide dismutase, catalase
and other free radical scavengers prevent this oxidative damage (45).
1.1.4 The malaria parasite genome
The malaria parasite is haploid throughout the life cycle, except for a brief time after
fertilization. Plasmodium falciparum has 5200 genes located on 14 chromosomes.
DNA is also present in the mitochondria and apicoplast. The apicoplast is transmitted
via the macrogamete. A large number of these genes are responsible for evasion of the
host immunity. The average gene density is approximately 1 gene / 4338 base pairs.
The mapping of this genome sequence provides new avenues for research on possible
vaccines. The genome of P. berghei, a rodent malaria parasite used in the evaluation
of antimalarias and vaccines, is highly similar, both in structure and gene content,
with P. falciparum (32).
1.1.5 Diagnosis of malaria
Diagnosis of malaria is based on clinical criteria (clinical diagnosis) supplemented by
the detection of parasites in the blood (parasitological or confirmatory diagnosis).
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Clinical diagnosis or symptomatic diagnosis involves evaluation of the clinical
presentation of the patient. The clinical presentation is based on if it is acute
uncomplicated malaria or acute severe malaria or chronic malaria. For acute
uncomplicated malaria, the clinical presentation includes; fever (temperature > 37.5
˚C, splenomegaly or hepatomegaly (especially in children), nail bed pallor, and the
patients history may include; subjective fever, chills and shivers, headache, tiredness,
body pains and joint weakness (46,47). The diagnosis of malaria based on clinical
symptoms alone is not reliable, as it results in unnecessary expenditure and
inappropriate use of antimalarial drugs, and a delay in establishing the correct
diagnosis and treatment objectives for a patient. 46 However, the manifestation of
malaria may range from asymptomatic to mild to severe disease such that the patient
can even present with only fever, due to the development of some level of immunity.
Consequently, it has been suggested that in stable malaria high-transmission areas like
Nigeria, the occurrence of fever (> 37.5 ˚C ) or a history of fever and no other obvious
cause, in children under 5 years and pregnant women also presenting with
unexplained pallor, suggests malaria, and treatment should be administered without
delay (46,47).
The clinical features of severe malaria includes; prostration, impaired consciousness,
respiratory distress, multiple convulsions (>2), shock, pulmonary oedema, abnormal
bleeding, jaundice, haemoglobinuria, renal failure, severe anemia and hypoglycemia.
Complications of severe malaria includes; cerebral malaria- a complication of severe
malaria caused by P. falciparum and it is malaria with coma persisting for greater
than 30 min after a seizure (46). Another complication of severe falciparum malaria is
black water fever, with features like; irregular fever, jaundice, dyspnea, intravascular
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hemolysis, renal failure, uremia and hemoglobinuria (coke coloured urine) (1, 46, 47).
Severe malaria is a medical emergency.
Laboratory diagnosis involves the use of microscopic examination of blood films and
antigen test or rapid diagnostic tests, RDTs. Since Charles Laveran first visualized the
malaria parasite in blood in 1880, (48) microscopic examination of blood films has
been the mainstay in the diagnosis of malaria. Its obvious advantage being that it is
quantitative nature. It involves the use of thin or thick blood smears that has been
stained with Romanowsky stain, and determination of the percentage parasitemia or
number of parasite per µl of blood respectively (24).
Thin films are similar to usual blood films and allow species identification of the
Plasmodium type, because the parasite's appearance is best preserved in this
preparation. Thick films allow the medical laboratory scientist to screen a larger
volume of blood and are about eleven times more sensitive than the thin film. Indeed
an experienced person can detect parasite levels (or parasitemia) down to as low as
0.0000001% of red blood cells. Therefore, picking up low levels of infection is easier
on the thick film, but the appearance of the parasite is much more distorted and
therefore distinguishing between the different species can be much more difficult.
With the pros and cons of both thick and thin smears taken into consideration, it is
imperative to utilize both smears while attempting to make a definitive diagnosis (49).
In severe malaria hyperparasitemia (i.e. a thin blood smear report of > 5% parasitemia
and a thick blood smear report of > 250,000 per µl of blood) is seen (47).
One important thing to note is that P. malariae and P. knowlesi (which is the most
common cause of malaria in South East Asia) look very similar under the microscope.
However, P. knowlesi parasitemia increases very fast and causes more severe disease
than P. malariae, so it is important to identify and treat infections quickly. Rapid
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Diagnostic Test and Molecular Methods should be used to distinguish between the
two in this area (50).
Rapid Diagnostic Test (or Antigen-Capture assay or Dip Stick Test) is a number of
immunochromatographic tests that make use finger-stick or venous blood, the
completed test takes a total of 15–20 minutes, and the results are read visually as the
presence or absence of colored stripes on the dipstick, so they are suitable for use in
the field. The threshold of detection by these rapid diagnostic tests is in the range of
100 parasites/µl of blood (commercial kits can range from about 0.002% to 0.1%
parasitemia) compared to 5 by thick film microscopy. Their disadvantage is that they
are qualitative but not quantitative. They also only differentiate falciparium malaria
from non-falciparium malaria; they cannot distinguish between other types of malaria
not caused by P. falciparium (24). The first rapid diagnostic tests were using P.
falciparum glutamate dehydrogenase as antigen (51). PGluDH was soon replaced by
P.falciparum lactate dehydrogenase, a 33 kDa oxidoreductase. It is the last enzyme of
the glycolytic pathway, essential for ATP generation and one of the most abundant
enzymes expressed by P. falciparum. PLDH does not persist in the blood but clears
about the same time as the parasites following successful treatment. The lack of
antigen persistence after treatment makes the pLDH test useful in predicting treatment
failure. In this respect, pLDH is similar to pGluDH. Depending on which monoclonal
antibodies are used, this type of assay can distinguish between all five different
species of human malaria parasites, because of antigenic differences between their
pLDH isoenzymes (50). RDTs are used in areas where microscopy is not available or
the laboratory staff is not experienced at malaria diagnosis (24).
A newer method of diagnosis called the molecular method involves the use of
Polymerase Chain Reaction (PCR) and QT-NASBA (based on the PCR) (52). PCR is
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more accurate than microscopy, but is more expensive and requires specialized
laboratories.
1.2 Prevention of malaria
Malaria can be prevented through prevention of its spread and protection of
individuals in endemic areas. Methods here include both environmental methods and
use of prophylactic treatment and includes; methods employed in mosquito
eradication, prevention of mosquito bites and use of prophylactic drugs. The
continued existence of malaria in an area requires a combination of high human
population density, high mosquito population density, and high rates of transmission
from humans to mosquitoes and from mosquitoes to humans. If any of these is
lowered sufficiently, the parasite will sooner or later disappear from that area, as
happened in North America, Europe and much of Middle East. However, unless the
parasite is eliminated from the whole world, it could become re-established if
conditions revert to a combination that favors the parasite's reproduction. Many
countries are seeing an increasing number of imported malaria cases due to extensive
travel and migration (50).
Brazil, Eritrea, India and Vietnam which are developing countries have successfully
reduced their malaria burden. Common success factors included conducive country
conditions, a targeted technical approach using a package of effective tools, data-
driven decision-making, active leadership at all levels of government, involvement of
communities, decentralized implementation and control of finances, skilled technical
and managerial capacity at national and sub-national levels, hands-on technical and
programmatic support from partner agencies, and sufficient and flexible financing
(53). Indeed, many researchers have argued that prevention of malaria is more cost-
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effective than treatment in the long run (54). Methods employed in mosquito
eradication encompass methods used in vector control and includes;
I. Draining of wetland breeding grounds.
II. Improved sanitation
III. Poisoning of breeding grounds of mosquitoes or aquatic habitats of the larva
stages, by filling or applying oil to stagnant waters.
IV. Researchers are going on to produce genetically modified mosquitoes that will
be malaria-resistant and then introduce them in the wild so that they gradually
replace the existing mosquitoes. This technique is called the sterile insect
technique. However, this approach contains many difficulties and success is a
distant prospect (55).
A futuristic method of vector control based, on the idea of using lasers to kill flying
mosquitoes (56).
Prevention of mosquito bites can be carried out by;
A. Indoor residual spraying (IRS): Here insecticides are sprayed on the interior
walls of homes in malaria affected areas. This is because after feeding many
mosquito species rest on nearby surface while digesting the blood meal, and if
the surface happens to be an insecticide sprayed wall, the mosquito will be
killed before they can take another blood meal (50). DDT was the first
pesticide used for this purpose (54). But, because it was also used in
agriculture and there was thus, emergence of mosquitoes resistant to DDT.
The use of DDT has now been banned or has limited use in agriculture for
some time now. Therefore, DDT may be more effective as a method of
malaria-control now (50). The WHO currently advises the use of 12 different
insecticides in IRS operations. These include DDT and a series of alternative
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insecticides (such as the pyrethroids, permethrin and deltamethrin), to combat
malaria in areas where mosquitoes are DDT-resistant and to slow the
evolution of resistance (57). This public health use of small amounts of DDT
is permitted under the Stockholm Convention on Persistent Organic Pollutants
(POPs), which prohibits the agricultural use of DDT. However, because of its
legacy, many developed countries discourage DDT use even in small
quantities (58). The problem with IRS is insecticide resistance which is
brought about by evolution. Mosquito species that are affected by IRS are
endophilic species (species that tend to rest and live indoors), and due to the
irritation caused by spraying, their evolutionary descendants are trending
towards becoming exophilic (species that tend to rest and live out of doors),
meaning that they are not as affected—if affected at all—by the IRS,
rendering it somewhat useless as a defense mechanism (59).
B. Use of mosquito nets and bedclothes: This involves the use of insecticide
treated nets (ITNs) that serve as repellants, thus keeping mosquitoes away
from people, and greatly reducing the infection and transmission of malaria.
The insecticide kills the mosquito before it has time to find a way through the
net and get to the individual. Even people sleeping near but not inside the net
are a bit protected. ITNs offer 70% protection compared with no net and are
twice as effective as untreated nets (60). However, less than 2% of children in
urban areas in sub-Saharan Africa are protected by ITNs. Insecticides
impregnated into the nets include; permethrin or deltamethrin. ITNs are the
most cost-effective method of malaria prevention (50). ITNs are to be re-
impregnated with insecticides every six months. Long-lasting Insecticidal nets
(LLINs), for example Olyset TM
, have now been produced and they release
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insecticides for approximately 5 years, but they cost more (50). Awareness on
the importance and appropriate use of ITNs has to be carried out.
A study carried out among Afghan refugees in Pakistan, has shown that top-sheets
and chadders (Head coverings) treated with permethrin has similar effectiveness as
ITNs, but is much cheaper (61).
Malaria prophylaxis involves the use of drugs to prevent the development of clinical
malaria, and can be in form of either intermittent preventive treatment (in pregnancy
or childhood) or chemoprophylaxis (in advantaged non-immune individuals travelling
to endemic areas). Most drugs used here are also used in the treatment of malaria, but
are taken at a lower dose than would be used for treatment, daily or weekly. Malaria
chemoprophylaxis is restricted to short term visitors and travelers to malaria endemic
regions, because of the adverse effects of long term use and cost of purchasing the
drug (50). Drugs used here includes; quinine, chloroquine, primaquine, quinacrine,
mefloquine, doxycycline, atovaquone + proguanil (malarone®).
There is currently no available effective vaccine. Malaria vaccines are still under
development and include: pre-erythrocytic vaccines (target parasites before it reaches
the blood), vaccines based on circum-sporozoite protein ( make up the largest group
of research for malaria vaccines), vaccines that seek to avoid more severe pathologies
of malaria by preventing adherence of the parasite to blood venules and placenta,
vaccines that seek to induce immunity to the blood stage of the infection, transmission
blocking vaccines ( it blocks the development of the parasite after a mosquito has
taken a blood meal from an infected person) (62). The first vaccine developed that has
undergone field trials, is the SPf66, developed by Manuel Elkin Patarroyo in 1987. It
presents a combination of antigens from the sporozoite (using CS repeats) and
merozoite parasites. During phase I trials a 75% efficacy rate was demonstrated and
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the vaccine appeared to be well tolerated by subjects and immunogenic. The phase IIb
and III trials were less promising, with the efficacy falling to between 38.8% and
60.2%. A trial was carried out in Tanzania in 1993 demonstrating the efficacy to be
31% after a year’s follow up, however the most recent (though controversial) study in
The Gambia did not show any effect. Despite the relatively long trial periods and the
number of studies carried out, it is still not known how the SPf66 vaccine confers
immunity; it therefore remains an unlikely solution to malaria. The CSP was the next
vaccine developed that initially appeared promising enough to undergo trials. It is also
based on the circumsporoziote protein, but additionally has the recombinant (Asn-
Ala-Pro15Asn-Val-Asp-Pro)2-Leu-Arg(R32LR) protein covalently bound to a
purified Pseudomonas aeruginosa toxin (A9). However at an early stage a complete
lack of protective immunity was demonstrated in those inoculated. The study group
used in Kenya had an 82% incidence of parasitaemia whilst the control group only
had an 89% incidence. The vaccine intended to cause an increased T-lymphocyte
response in those exposed; this was also not observed (62).
The RTS, S/AS02A vaccine is the candidate furthest along in vaccine trials. It is being
developed by a partnership between the PATH Malaria Vaccine Initiative (a grantee
of the Gates Foundation), the pharmaceutical company, GlaxoSmithKline, and the
Walter Reed Army Institute of Research (63). In the vaccine, a portion of CSP has
been fused to the immunogenic "S antigen" of the hepatitis B virus; this recombinant
protein is injected alongside the potent AS02A adjuvant (62). In October 2004, the
RTS, S/AS02A researchers announced results of a Phase II b trial, indicating the
vaccine reduced infection risk by approximately 30% and severity of infection by
over 50%. The study looked at over 2,000 Mozambican children (64). More recent
testing of the RTS, S/AS02A vaccine has focused on the safety and efficacy of
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administering it earlier in infancy: In October 2007, the researchers announced results
of a phase I/IIb trial conducted on 214 Mozambican infants between the ages of 10
and 18 months in which the full three-dose course of the vaccine led to a 62%
reduction of infection with no serious side-effects save some pain at the point of
injection (65).
Other methods of prevention of malaria includes; mass drug administration,
intermittent preventive therapy, education of the populace on preventive methods, and
providing accessible malaria diagnostic facilities and affordable, accessible, effective
drugs. More so, screening of windows and doors with mosquito netting, and use of
mosquito repellant cream can protect against malaria infection (66, 24).
1.3 Treatment of malaria
Historically, the first effective treatment for malaria came from the bark of cinchona
tree, which contains quinine. This tree grows on the slopes of the Andes, mainly in
Peru. The indigenous peoples of Peru made a tincture of cinchona to control malaria.
The Jesuits noted the efficacy of the practice and introduced the treatment to Europe
during the 1640s, where it was rapidly accepted (67). It was not until 1820 that the
active ingredient, quinine, was extracted from the bark, isolated and named by the
French chemists Pierre Joseph Pelletier and Joseph Bienaimé Caventou (68). Due to
the toxicity of quinine, a less toxic analoque, chloroquine was developed and was
designated the drug of choice in 1946 (69). Other more effective and less toxic
antimalarials have been synthesized since then.
The therapy for malaria differs based on if the malaria is uncomplicated or severe.
Uncomplicated malaria can be treated at home while severe malaria is an emergency
and is treated in the hospital. Whether uncomplicated or not, supportive
measures/treatment must be added in addition to the specific antimalarial drug used.
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The antimalarial drug can be inform of approved herbal remedies, as is most preferred
in the rural areas of developing countries due to the high cost of orthodox antimalarial
drugs (50). The WHO guidelines for the treatment of malaria is presented in section
1.6 below and it provides a protocol on how the drugs discussed here are to be used in
the treatment of uncomplicated malaria.
1.3.1 Drugs used in the treatment of malaria
Chemotherapy is the prevention or treatment of a disease through the use of chemical
substances (70). From the chemotherapeutic standpoint, antimalarial agents are
classified into four main groups;
i. Tissue schizontocides- Antimalarial agents that are tissue schizontocides act
on the developing or dormant forms of the parasite in the hepatocytes (71),
and so will prevent relapse (fresh infection) from P. vivax and P. ovale,
Examples of tissue schizontocides are proguanil and pyrimethamine (1). They
are also used in chemoprophylaxis.
ii. Blood schizontocides- Antimalarial agents belonging to this group act on the
erythrocytic asexual forms of the parasite. They thus prevent the manifestation
of clinical disease, thus producing a cure. Majority or all of effective
antimalarial drugs kill the erythrocytic form of the parasite, and thus reduce
the number of blood forms before they grow in sufficiently large quantities to
cause clinical disease (71).
iii. Gametocytocide- Antimalarial agents in this group act on the sexual forms
(gametocytes) of the parasite in the blood, which is responsible for the
continual transmission of malaria by mosquitoes. Some gametocytocides act
on the early forms of the gametocytes, while others act on all stages of
maturation. Different gametocytocides are known to demonstrate varying
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effects on the gametocytes of the different species of Plasmodium. Only
primaquine is known to act on the gametocytes of all species (1).
iv. Sporontocides- The antimalarial agents in this group render the gametocytes
infertile, instead of killing them, by inhibiting the sporogonic cycle in the
mosquito that has sucked the infected blood from a person treated with
sporontocides. An example of sporontocides is pyrimethamine (1).
Based on their function or the way they are used, antimalarial agents can be classified
as;
i. Antimalarial agents used for causal prophylaxis: Causal prophylaxis is the
process by which the infection is controlled before symptoms of the disease
begin to manifest. Therefore an agent used for causal prophylaxis must act on
the hepatic forms of the parasite, thus eradicating the parasite before they
reach the blood stream. P. falciparium is the most susceptible to antimalaria
prophylactic treatment. Examples of antimalarial agents used for causal
prophylaxis are proguanil and primaquine, which is more active than the
former (1).
ii. Antimalarial agents used for suppressive treatment: the drugs in this group act
on the erythrocytic forms of the parasite, thus suppressing the symptoms of
malaria. Symptoms of the malaria may return if these agents are taken for a
short period due to the hepatic stage of the parasite still in the liver, which may
emerge and enter into the blood. But if the drug is taken for a longer period a
cure may be achieved. An example of such drugs is pyrimethamine (1).
iii. Antimalarial agents used for clinical cure: drugs in this group are active
against the asexual erythrocytic stages, thus preventing the development of
schizonts. Examples of drugs here are; chloroquine, amodiaquine, quinacrine,
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quinine, mefloquine, halofantrine, artemisinin derivatives, atovaquone,
sulfedoxine +pyrimethamine etc (1).
iv. Antimalarial agents used for radical cure: drugs in group can eradicate both
the exoerythrocytic and the erythrocytic stages of the parasite, but no single
drug can reliably produce radical cure (71), the drugs have to be used in
combination. Examples include; primaquine + chloroquine, primaquine +
quinine etc
v. Miscellaneous Antimalarial agents: this includes antibiotics that have been
shown to have antimalaria activity either in vitro or in vivo. The most widely
used antibiotics are the tetracyclines, doxycycline and minocyclin, because of
their activity on both the exoerythrocytic schizonts and the asexual forms in
the blood (1). They are not used alone in the treatment of malaria; they are
combined with drugs used for clinical cure.
Combinations potentially offer a number of important advantages over
monotherapies. First, they should provide improved efficacy. Appropriately chosen
combinations must be at least additive in potency, and may provide synergistic
activity. However, combination regimens that rely on synergy may not offer as much
protection against the selection of resistance as expected, as resistance to either
component of the combination could lead to a marked loss of efficacy. Indeed, the
widely used synergistic combination SP acts almost as a single agent in this regard,
with rapid selection of resistance (72), and similar concerns apply to the new
atovaquone/proguanil (Malarone; GlaxoSmithKline) combination (73). Other
desirable properties of antimalaria combination are that; resistance should not develop
to the two drugs at a time and the combination should reduce the selection of
antimalaria drug resistance. It was also recently shown that SP selected for resistance-
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conferring mutations and subsequent treatment failure, but that SP combined with
artesunate prevented the selection of SP-resistant parasites in subsequent infections
(72). Combinations might offer additional advantages if the separate agents are active
against different parasite stages and if they provide the opportunity to decrease
dosages of individual agents, thereby reducing cost and/or toxicity. Ideally,
combination regimens will incorporate two agents that are both new (so that parasites
resistant to either agent are not already circulating), offer potent efficacy and
preferably have similar pharmacokinetic profiles (to limit the exposure of single
agents to resistance pressures). Unfortunately, these are challenging requirements that
are not met by any combination available at present. One current, widely advocated
strategy is to combine artemisinins — which have no resistance problem but suffer as
monotherapy from late recrudescences due to their short half-lives (71) with longer-
acting agents. The hope is that the potent action of artemisinins will prevent
significant selection of parasites resistant to the longer-acting component like
amodiaquine, mefloquine and lumefantrin etc (74).
1.3.2 Artesunate
Artesunate is a semisynthetic derivative of artemisinin (a potent antimalaria drug
isolated from Artemisia annua) that was developed so as to address the problems
encountered during the formulation of artemisinin into oral dosage forms, via
improving the solubility of the parent drug (71). Other analogues include artemether,
dihydroartemisinin etc. Artemisinin and its derivatives have superior antimalarial
efficacy (fever clearance time is shortened to 32 h as compared with 2-3 days with
older drugs (75)), are effective in the treatment of severe malaria (comparable to that
of quinine), and is effective in cases of multi-drug resistant falciparium malaria
(1,71). They are even effective against quinine resistant strains.
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Although their efficacy is limited by their short half-life, such that recrudescence rates
are unacceptably high after a short course or even after a seven day therapy, efficacy
can be maintained by combining them with other antimalarial like mefloquine,
lumefantrine, fansider etc. These combinations also curb resistance. They are also
safer than quinine and have lesser adverse effects compared to quinine (1,71). It is
soluble in water but has poor stability in aqueous solutions at neutral or acid pH. In
the injectable form, artesunic acid is drawn up in sodium bicarbonate to form sodium
artesunate immediately before injection.
Figure 2: Artesunic acid
1.3.2.1 Mechanism of action of artesunate
Artesunate is converted rapidly in the body into mainly dihydroartemisinin (DHA),
the active metabolite of artesunate, by the plasma esterases. Dihydroartemisinine
(DHA) like artesunate has an endoperoxide bridge, as shown in Figure 2. This
endoperoxide bridge is split by heme or molecular iron within the infected
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erythrocyte, generating singlet oxygen or free radicals and electrophilic (alkylating)
intermediates. Parasite proteins, particularly in membraneous structures, are thus
akylated, leading to parasite death. They are active against a broad spectrum of the
life cycle of the parasite, from the relatively inactive ring stage to the late schizonts in
the blood cells. The schizontocidal and gametocytocidal activities of artesunate
administered orally have been demonstrated in vivo on chloroquine sensitive stains of
P. berghei in mice and P. knowlesi in monkey) and chloroquine resistant strains of P.
berghei in mice (76).
1.3.2.2 Pharmacokinetics of artesunate
Artesunate is given orally, intraveneously, intramuscularly and rectally. It is rapidly
absorbed after oral administration and then rapidly converted by plasma esterases to
mainly DHA, with peak plasma concentration at 1.5 hrs post administration, and a
half-life of 45 min. DHA drug levels appear to decrease after a number of days of
therapy. DHA is highly protein bound. DHA is then metabolized in the liver via
glucuronidation prior to excretion (46, 71).
1.3.2.3 Pharmacological actions of artesunate
After administration artesunate acts very rapidly against blood schizontocides of all
species of human plasmodium, more rapidly than even oral chloroquine or
intravenous quinine, but has no effect on the hepatic stage. Artemisinins have
gametocytocidal effects (71). Artesunate is indeed the fastest acting artemisinin used
clinically (77).
1.3.2.4 Side effects of artesunate
Artemisinins and its derivatives are better tolerated than most antimalarias. Side
effects are few and includes; nausea, vomiting, diarrhea, rarely; transient
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reticulocytopenia, neurological abnormalities and some liver enzyme elevation. At
doses higher than those used to treat malaria irreversible neurotoxicity occur (76).
1.3.2.5 Artemisinin-based combination therapy
As in HIV management, the World Health Organization (WHO) guidelines now
recommend antimalarial combination therapy to forestall the development of further
resistance (46). Combination therapy of antimalarial drug refers to the simultaneous
use of two or more blood schizontocidal drugs with independent mode of action and
different biochemical targets in the parasites. The concept of combination therapy is
based on the synergistic or additive potential of two or more drugs to improve
therapeutic efficacy and also to delay the development of resistance to the individual
components of the combination (78). Combination
therapy should include a
gametocidal agent to decrease transmission
of both sensitive and drug-resistant
parasites (79). The effect of combination therapy is enhanced by the inclusion of an
artemisinin derivative, as they decrease the parasite density more rapidly than other
antimalaria drug (80). When used alone, the short half-life of the artemisinin
derivative minimizes the period of parasite exposure to subtherapeutic blood levels. In
combination with another drug with a longer half-life, the short half-life and rapid
parasite clearance time of artemisinin derivatives mean that fewer parasites are
exposed to the companion drug alone after elimination of the artemisinin component.
Furthermore, exposure occurs when blood levels of the drug close to the maximum
are still present (81).
Another benefit of artemisinin combination is the 90% reduction in gametocyte levels
in treated patients (82). These characteristics minimizes the probability that a resistant
mutant will survive therapy and may also reduce the overall malaria transmission
rates, especially via pre-existing resistant strains. Artemisinin-combination therapies
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(ACTs) are WHO-recommended treatment policy for uncomplicated malaria (83) in
countries where standard antimalarials are ineffective due to drug resistance (79). The
use of this drug alone results in recrudescence because of its short half-life; therefore,
it must be used in combination. In Africa, artemeter-lumefantrine is recommended as
the standard combination therapy, but other combination therapy have been studied
and are used (84,85). Polymorphisms within the pfatp6 gene have been associated
with the resistant phenotype is some studies. It may be of concern that pfmdr1
mediates resistance to a number of unrelated classes of agents that are now being used
in combination (86). Continued monitoring of genotypic, phenotypic, and in vivo
outcomes will be necessary to monitor efficacy of these new combinations.
Current
combinations
recommended by the WHO include artemether-lumefantrine,
artesunate-amodiaquine, artesunate-mefloquine, and artesunate-SP, for second line
treatment; alternative ACT or quinine in combination with either tetracycline or
doxycycline or clindamycin (46).
1.4 Pharmacodynamic interaction
Pharmacodynamic interactions include the concurrent administration of drugs having
the same (or opposing) pharmacologic actions and alteration of the sensitivity or the
responsiveness of the tissues to one drug by another. Many of these interactions can
be predicted from knowledge of the pharmacology of each drug (87). When
discussing drug interactions, the drug affected by the interaction is called the “object
drug,” and the drug causing the interaction is called the “precipitant drug.”(88). It
involves competition at receptor sites or activity of the interacting drugs on the same
physiological system.
Types of pharmacodynamic drug interaction include;
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A. Additive/ synergistic pharmacodynamic drug interaction: When two or more
drugs with similar pharmacological actions are given, the combined effect
may be additive or synergistic, i.e., an effect that is more than the singular
effect of the individual drugs. This can result in excessive response and
toxicity. Examples include combinations of alcohol and hypnosedatives,
which may result in excessive drowsiness; potassium supplements and
potassium-sparing diuretics, which will cause marked hyperkalaemia.
B. Antagonistic pharmacodynamic drug interaction: Here drugs with opposing
pharmacological actions acting on the same receptor. The response to one or
both drugs may be reduced. For example, drugs that tend to increase blood
pressure (such as nonsteroidal anti-inflammatory drugs) may inhibit the
antihypertensive effect of drugs such as ACE inhibitors.
C. Pharmacodynamic interaction due to fluid/electrolyte imbalance:
E.g. diuretics that cause hypokalaemia can increase the toxicity of digoxin.
D. Pharmacodynamic interaction due to changes in drug transport mechanisms:
chlorpromazine, when given together with guanethidin, an antihypertensive,
will inhibit the uptake of guanethidine into the adrenergic neurons, thus
inhibiting its antihypertensive effect (87).
1.5 Antimalarial drug resistance
Drug resistance is reduced susceptibility of the causal agent of a disease to a drug.
According to WHO, antimalarial drug resistance is the ability of a parasite strain to
survive and/or multiply despite the administration and absorption of a medicine given
in equal to or higher than those usually recommended but within the tolerance of the
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subject, with certainty that the form of the drug active against the parasite will gain
access to the parasite or the infected red blood cell for the duration of time necessary
for its normal action in the body. Resistance arises due to selection of parasites with
genetic mutations or gene amplifications that confer reduced susceptibility (46).
Efforts to eradicate or control the disease, by eradicating the female mosquito via the
use of insecticides or by therapy with antimalarials have proved abortive, especially in
developing nations due to the emergence of insecticide resistant mosquito and drug-
resistant malaria parasites (78). The high prevalence of infection, constant drug
pressure, sexual reproduction, and large parasite biomass
contribute to the
development of a resistant parasite. These factors present a unique challenge in
optimizing drug treatment strategies. This scenario is similar to HIV, in which
continuous drug pressure and high viral burden have resulted in drug-resistant
strains.
As in HIV management, the World Health Organization (WHO) guidelines now
recommend antimalarial combination therapy to forestall the development of further
resistance (46). The United Nations International Children’s’ Education Fund
(UNICEF) notes that the greatest challenge in malaria control is that the cheapest
antimalarial drug chloroquine is rapidly losing its effectiveness, via the development
of resistance (1). Gametocytes are often resistant to standard antimalarials used to
clear the asexual-stage parasite. This may be due to the
unique biology of
gametocytes, which shuts down a subset of
metabolic pathways, rendering the
antimalarials ineffective (89). The post treatment gametocyte carriage rate varies by
antimalarial treatment, and in vitro evidence suggests that chloroquine may
actually
induce gametocytogenesis (90,91,92). The artemisinin compounds
are a notable
exception and can clear gametocyte stages.
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The history of antimalarial drug resistance dates back to 1963. This resistance did not
appear when pure single compounds were used, most probably because the herbal
remedies used to treat malaria were already in form of a complex mixture, with
numerous antimalarial agents acting at the same time thus making it difficult for the
parasite to develop resistance against a single agent or even all the agents at the same
time. Indeed, combination therapy existed in herbal remedies from time immemorial.
Chloroquine was designated the drug of choice in 1946 (69). In Malaysia, chloroquine
resistant case was first reported in 1963 (93). Subsequently, several Chloroquine-
resistant cases have been reported in Sabah, west Malaysia (94). In Africa,
chloroquine resistant Plasmodium falciparum was first found in 1978 in nonimmune
travellers from Kenya and Tanzania (95, 96). This was followed 2 to 3 years later by
reports from Madagascar (97). Resistance spread from the African coastal areas inland
and by 1983 had been observed in Sudan, Uganda, Zambia and Malawi (98, 99, 100,
101). The Sulphonamides-Pyrimethamine (SP) combination was not left out
(102,103,104,105,106). Recently, there has been wide spread resistance of
falciparium malaria both to chloroquine and SP in endemic areas of Peninsular
Malaysia (107). In Thailand, SP replaced chloroquine in 1973 due to resistance, and
then mefloquine-sulphadoxine-pyrimethamine (MSP) was introduced in 1984 in the
Thai-cambodian border refugee camp and later extended to the whole of Thailand in
1985. In 1989, there was a reduction in the sensitivity (95% to 50%) to mefloquine
(MQ) at 15mg/kg, in specific areas of Thailand (108). MQ at 25mg/kg was even still
unable to prolong higher efficacy substantially, so that the use of MQ plus 3 days of
artesunate (an ACT) was adopted in such areas from 1994-1996, and this restored the
cure rate to 90-95% (109). In, Africa, efficacy trials have also shown the superiority of
artemisinin combination therapy when compared with monotherapy in areas of drug-
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resistant malaria
(110,111). Moreover, the therapeutic failure of artesunate
monotherapy in non-immune individuals from Central Africa were associated with
reduced in vitro susceptibility (112,113), but, there is no confirmed in vivo evidence
of resistance of P. falciparum to artemisinin and its derivatives.
Methods for assessing and thereby classifying antimalarial drug resistance include;
A. In vivo Testing: This involves the assessment of clinical and parasitological
outcomes of treatment over a certain period (≥ 28 days) following the start of
treatment, to check for the reappearance of parasites in the blood.
Reappearance indicates reduced parasite sensitivity to the treatment drug. The
parasitological cure rates should also be assessed. Blood or plasma of the
antimalaria drug has to be assessed also so as to distinguish treatment failures
due to pharmacokinetic reasons (46).
B. Molecular genotyping: this involves the use of molecular markers for drug
resistant malaria. These markers are then detected by using PCR, where small
amounts of parasite DNA material in finger-prick blood dried on filter paper
are used. These markers are based on genetic changes that confer parasite
resistance to drugs used to treat and prevent malaria. Polymorphisms in the
Plasmodium falciparum chloroquine resistance transporter (PfCRT) confer
resistance to chloroquine (114,115), and mutations in the P-glycoprotein
homologue (Pgh1) encoded by pfmdr1 modulate this resistance (116).
Polymorphisms in pfmdr1 and amplifications of this gene also affect
susceptibility to structurally unrelated antimalarial drugs, including
mefloquine, artesunate, lumefantrine and quinine (117,118,119).
Polymorphisms in P. falciparum dihydrofolate reductase (DHFR) cause
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resistance to the antifolate drugs including pyrimethamine and other DHFR
inhibitors, and polymorphisms in dihydropteroate synthase (DHPS) cause
resistance to sulphadoxine and other sulphas and sulphones (120,121). For
ACT, molecular markers are to be used to monitor its partner drug. Each ACT
partner drug is likely to select for resistance, potentially leading to loss of
treatment efficacy as well as failure to protect the artemisinins against the
development of resistance. At present the most important partner drugs used
with artemisinins in ACTs are amodiaquine, lumefantrine, and piperaquine.
The molecular mediators of resistance are not as well defined for these drugs
as they are for chloroquine and SP, but recent data show hints of mechanisms
of resistance. For amodiaquine, polymorphisms in both PfCRT and Pgh1
appear to predict resistance and to be selected for by treatment with
amodiaquine (122) or artesunate-amodiaquine (123). Artemether-lumefantrine
treatment selects for polymorphisms in pfmdr1 associated with diminished
sensitivity to the related drug halofantrine (124,125). Markers for piperaquine
resistance have not been identified, but this aminoquinoline may well act
similarly to chloroquine and amodiaquine in its selection of resistance-
mediating mutations. To avoid unacceptably long delays in identifying,
validating and deploying molecular markers of ACT resistance, and the
malaria research and control community must be prepared to investigate
aggressively early reports of resistance, confirm resistance with careful in
vitro assays, and bring genetic and genomic tools to bear to elucidate
mechanisms and identify candidate molecular markers. The sequencing of the
P. falciparum genome has led to genome-wide approaches that may help to
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identify genetic markers of drug resistance far more quickly than was
previously possible (126,127,128).
There is a network database being created, that will be used to monitor and
deter resistance, and to guide malaria treatment and prevention policies called
World Antimalaria Resistance Network (WARN), which uses a global
database for molecular markers of drug resistant malaria and links it to
databases for malaria drug efficacy trials, in vitro drug resistance, and
pharmacokinetics (129). Molecular genotyping using PCR technology should
be used to distinguish recrudescent parasites from newly acquired infections.
C. In vitro testing: It involves the collection of parasitized blood from patients
and the testing of parasite susceptibility to drugs in culture. Here the most
commonly used methods are the in vitro micro-test Mark III the isotopic test
and drug sensitivity assay based on the measurement of HRP2/or pLDH/ in an
enzyme-linked immunosorbent assay (ELISA). To support evidence of a
failing antimalarial, in vitro tests can be used to provide a more accurate
measure of drug sensitivity under controlled experimental conditions.
Parasites obtained from finger-prick blood are placed in microtitre wells,
exposed to precisely known concentrations of a particular drug and examined
for the inhibition of maturation into schizont parasite stages (130). This test
overcomes some of the many confounding factors influencing the results of in
vivo tests, such as subtherapeutic drug concentrations and the influence of host
factors on parasite growth (e.g. factors related to acquired immunity), and
therefore provide a more accurate picture of the “true” level of resistance to
the drug. Multiple tests can be performed on parasite isolates, using several
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drugs and drug combinations. But only P. falciparium and P. vivax can be
tested using this method. In vitro testing is more demanding in terms of
technology and resources, and is not ideal for routine drug efficacy evaluation
under field conditions. It should therefore primarily be used to provide
additional information to support clinical efficacy data at selected resistance-
monitoring sites.
1.6 WHO Guildlines for treatment of uncomplicated malaria
For several decades, the gold standard for the treatment of malaria was chloroquine, a
4-aminoquinoline (it was efficacious, had low toxicity and was affordable (74). It now
recommended that the first line treatment for malaria in any region be changed if the
total failure proportion exceeds 10%. However, it is acknowledged that a decision to
change may be influenced by a number of additional factors, including the prevalence
and geographical distribution of reported treatment failures, health service provider
and/or patient dissatisfaction with the treatment, the political and economical context,
and the availability of affordable alternatives to the commonly used treatment. To
overcome the threat of resistance of P. falciparum to monotherapies, and to improve
treatment outcome, combinations of antimalarials are now recommended for the
treatment of uncomplicated falciparum malaria. Drug combinations such as
sulfadoxine–pyrimethamine, sulfalene–pyrimethamine, proguanil-dapsone,
chlorproguanil-dapsone and atovaquone-proguanil rely on synergy between the two
components. The drug targets in the malaria parasite are linked. These combinations
are operationally considered as single products and treatment with them is not
considered to be antimalarial combination therapy. Multiple-drug therapies that
include a non-antimalarial medicine to enhance the antimalarial effect of a blood
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schizontocidal drug (e.g. chloroquine and chlorpheniramine) are also not antimalarial
combination therapy.
The rationale for combining antimalarials with different modes of action is twofold:
(1) the combination is often more effective; and (2) in the rare event that a mutant
parasite that is resistant to one of the drugs arises de novo during the course of the
infection, the parasite will be killed by the other drug. This mutual protection is
thought to prevent or delay the emergence of resistance.
To realize the two advantages, the partner drugs in a combination must be
independently effective. The possible disadvantages of combination treatments are the
potential for increased risk of adverse effects and the increased cost.
Artemisinin and its derivatives (artesunate, artemether, artemotil, dihydroartemisinin)
produce rapid clearance of parasitaemia and rapid resolution of symptoms. They
reduce parasite numbers by a factor of approximately 10000 in each asexual cycle,
which is more than other current antimalarials (which reduce parasite numbers 100- to
1000-fold per cycle). Artemisinin and its derivatives are eliminated rapidly. When
given in combination with rapidly eliminated compounds (tetracyclines, clindamycin),
a 7-day course of treatment with an artemisinin compound is required; but when given
in combination with slowly eliminated antimalarials, shorter courses of treatment (3
days) are effective. In 3-day ACT regimens, the artemisinin component is present in
the body during only two asexual parasite life-cycles (each lasting 2 days, except for
P. malariae infections). This exposure to 3 days of artemisinin treatment reduces the
number of parasites in the body by a factor of approximately one hundred million (104
× 104 =10
8). However, complete clearance of parasites is dependent on the partner
medicine being effective and persisting at parasiticidal concentrations until all the
infecting parasites have been killed. Thus the partner compounds need to be relatively
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slowly eliminated. As a result of this the artemisinin component is “protected” from
resistance by the partner medicine provided it is efficacious and the partner medicine
is partly protected by the artemisinin derivative. Courses of ACTs of 1–2 days are not
recommended; they are less efficacious, and provide less protection of the slowly
eliminated partner antimalarial. The artemisinin compounds are active against all four
species of malaria parasites that infect humans and are generally well tolerated. The
only significant adverse effect to emerge from extensive clinical trials has been rare
(with an occurrence of 1:3000) and is type 1 hypersensitivity reactions (manifested
initially by urticaria). The artemisinins also have the advantage of reducing
gametocyte carriage and thus the transmissibility of malaria. This contributes to
malaria control in areas of low endemicity.
Non-artemisinin based combinations (non-ACTs) include sulfadoxine–pyrimethamine
with chloroquine (SP+CQ) or amodiaquine (SP+AQ). However, the prevailing high
levels of resistance have compromised the efficacy of these combinations. There is no
convincing evidence that SP+CQ provides any additional benefit over SP, so this
combination is not recommended; SP+AQ can be more effective than either drug
alone, but needs to be considered in the light of comparison with ACTs.
The following ACTs are currently recommended (alphabetical order);
artemether-lumefantrine,
artesunate + amodiaquine,
artesunate + mefloquine,
artesunate + sulfadoxine–pyrimethamine.
Amodiaquine plus sulfadoxine–pyrimethamine may be considered as an interim
option where ACTs cannot be made available, provided that efficacy of both is high.
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The following drugs have not yet been recommended as an antimalarial-combination
therapy;
Chlorproguanil-dapsone has not yet been evaluated as an ACT partner drug, so
there is insufficient evidence of both efficacy and safety to recommend it as a
combination partner.
Atovaquone-proguanil has been shown to be safe and effective as a
combination partner in one large study, but is not included in these
recommendations for deployment in endemic areas because of its very high
cost.
Halofantrine has not yet been evaluated as an ACT partner medicine and is not
included in these recommendations because of safety concerns.
Dihydroartemisinin (artenimol)-piperaquine has been shown to be safe and
effective in large trials in Asia, but is not included in these recommendations
as it is not yet available as a formulation manufactured under good
manufacturing practices, and has not yet been evaluated sufficiently in Africa
and South America.
Treatment failure within 14 days of receiving an ACT is very unusual. Treatment
failures within 14 days should be treated with a second-line antimalarial;
alternative ACT known to be effective in the region,
artesunate + tetracycline or doxycycline or clindamycin,
quinine + tetracycline or doxycycline or clindamycin.
Recurrence of fever and parasitaemia more than 2 weeks after treatment, which could
result either from recrudescence or new infection, can be retreated with the first-line
ACT. Parasitological confirmation is desirable but not a precondition. If it is a
recrudescence, then the first-line treatment should still be effective in most cases. This
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simplifies operational management and drug deployment. However, reuse of
mefloquine within 28 days of first treatment is associated with an increased risk of
neuropsychiatric sequelae and, in this particular case; second-line treatment should be
given. If there is a further recurrence, then malaria should be confirmed
parasitologically and second-line treatment given.
In pregnancy, the antimalarials considered safe in the first trimester of pregnancy are
quinine, chloroquine, proguanil, pyrimethamine and sulfadoxine–pyrimethamine. Of
these, quinine remains the most effective and can be used in all trimesters of
pregnancy. In reality women often do not declare their pregnancies in the first
trimester and so, early pregnancies will often be exposed inadvertently to the
available first-line treatment. Inadvertent exposure to antimalarials is not an indication
for termination of the pregnancy. There is increasing experience with artemisinin
derivatives in the second and third trimesters (over 1000 documented pregnancies).
There have been no adverse effects on the mother or fetus. The current assessment of
benefits compared with potential risks suggests that the artemisinin derivatives should
be used to treat uncomplicated falciparum malaria in the second and third trimesters
of pregnancy, but should not be used in the first trimester until more information
becomes available. The choice of combination partner is difficult. Mefloquine has
been associated with an increased risk of stillbirth in large observational studies in
Thailand, but not in Malawi. Amodiaquine, chlorproguanil-dapsone, halofantrine,
lumefantrine and piperaquine have not been evaluated sufficiently to permit positive
recommendations. Sulfadoxine–pyrimethamine is safe but may be ineffective in many
areas because of increasing resistance. Clindamycin is also safe, but both medicines
(clindamycin and the artemisinin partner) must be given for 7 days. Primaquine and
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tetracyclines should not be used in pregnancy. Dapsone and tetracycline are not to be
used by lactating mothers.
P. malariae and P. ovale are still very sensitive to chloroquine, and are also sensitive
to amodiaquine, mefloquine and the artemisinin derivatives. P. vivax is still very
sensitive to chloroquine but resistance is prevalent and increasing in some areas,
notably Oceania, Indonesia and Peru. It is also sensitive to all other antimalarias
except SP, proguanil and chlorproguanil. In contrast to P. falciparum, asexual stages
of P.vivax are susceptible to primaquine. Thus the combination of chloroquine and
primaquine can be considered a combination treatment. The only drugs with
significant activity against the hypnozoites are the 8-aminoquinolines (bulaquine,
primaquine, tafenoquine). The recommended treatment for the relapsing malaria
caused by P. ovale is the same as that given to achieve radical cure in vivax malaria,
i.e. with chloroquine and primaquine. Mixed infections are common and ACTs are
effective against all malaria species, and so is the treatment of choice. Radical
treatment with primaquine should be given to patients with confirmed P. vivax and P.
ovale infections except in high transmission settings where the risk of re-infection is
high (46).
1.7 Plants with antimalarial activity
Plants remain an important source of medicines for both traditional and orthodox
health care practices. For example, artemisinin, which was isolated from Artemisia
annua L. (Qinghaosu), the sweet wormwood, and is a sesquiterpene lactone
endoperoxide (1). Traditionally, plants are used in form of herbal drugs or
phytomedicines. Over the past century, chemical and pharmacologic science
established the compositions, biological activities and health giving benefits of
numerous plant extracts. But often when individual components were separated from
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the whole there was loss of activity—the natural ingredient synergy became lost.
Standardization was developed to solve this problem. But even with standardization,
poor bioavailability often limited their clinical utility (131). In 2001, researchers
identified 122 compounds used in mainstream medicine which were derived from
ethnomedical plant sources; 80% of these compounds were used in the same or
related manner as the traditional ethnomedical use. Major pharmaceutical companies
are currently conducting extensive research on plant materials gathered from the rain
forests and other places for possible new pharmaceuticals (132). Three quarters of
plants that provide active ingredients for prescription drugs came to the attention of
researchers because of their use in traditional medicine. Among the 120 active
compounds currently isolated from the higher plants and widely used in modern
medicine today, 75 percent show a positive correlation between their modern
therapeutic use and the traditional use of the plants from which they are derived (132).
More than two thirds of the world's plant species - at least 35,000 of which are
estimated to have medicinal value - come from the developing countries. At least
7,000 medical compounds in the modern pharmacopoeia are derived from plants
(133).
In Indonesia’s malaria endemic regions, medicinal plants such as Carica papaya
leaves, Eurycoma longifolia, Alstonia scolaris, Phyllanthus niuriri and Azadirachta
indica are often used to treat malaria. However, scientific information on the
antimalarial activity of these plants is very limited. Artemisia annua and Azadirachta
indica are considered as reference medicinal plants by numerious author due to their
wide use traditionally, in the treatment of malaria (134). Key medicinal plants used by
ethnomedicinal practitioners in the treatment of malaria in Nigeria are; Venonia
amygdalina, Ageratum conyzoides and Azadirachta indica (135). Most commonly
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used plant for the treatment of malaria in Southwest, South south and Middle Belt are;
A. indica, Cymbopogon citrates and Carica papaya (136). The following herbs have
also shown some antimalarial activity in vitro or in animals: Artemisia vulgaris,
Cochlospermum planchonii, Cochlospermum tinctorium, Jatropha curcas, Gossypium
hirsutum, Physalis angulata, Delonix regia, Khaya grandifolia, Cryptolepsis
sanguinolenta, Tabebuia impetiginosa, Carica papaya, Swertia chirayita,
Azadirachta indica, Cajanus cajan, Euphorbia lateriflora, Mangifera indicia, Senna
alata, Cymbopogon giganteus, Nauclea latifolia, Newbouldia leavis, Adansonia
digitata, Cassia occidentalis, Tamarindus indica, Tridax procumbens, Vernonia
amygdalina, Psidium guajava, Morinda lucida and Uvaria chamae etc.
Phytomedicines initially made it possible to reliably treat malaria (via the use of
quinine from Cinchona spp.) and continue to provide exciting new antimalarial drugs
(the discovery of the artemisinins from A. annua).
1.7.1 Antimalaria activity of Carica papaya
The male and female parts of Carica papaya Linn. (Caricaceae; Common Name:
pawpaw) exist in different trees. The fruits, leaves, and latex are used medicinally.
The taxonomy of Carica papaya is shown below;
Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Order: Violales
Family: Caricaceae
Genus: Carica L.
Species: Carica papaya L.
Its mature leaves are widely used to treat malaria and splenomegaly while the fruit is
used against anaemia, which can also be caused by malaria (137). The anti-plasmodial
activity of Carica papaya is weak as it has an IC50 of 60 mg/mL (138). However, a
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recent study has shown it to reduce parasitemia at an activity second to that of SP.
Aqueous extract of its leaves have been shown to reduce parasite count from 9.20 ±
0.06 to 2.60 ± 0.06 % in P. berghei infected mice (139). In Ghana, pawpaw leaves,
neem leaves/bitter leaves, boiled together in water and cooled is taken at a dosage of
30 ml taken 3 x daily x 7 days. In Nigeria, a weak decoction of the leaves is taken for
the treatment of malaria (140).
Its antimalarial activity has been attributed to its ability to increase total antioxidant
status in patients and thus, inhibiting the development of anemia in malaria (141).
1.7.2 Antimalaria activity of Azadirachta indica
Azadirachta indica juss. (Common Name: neem) is an evergreen tree, but in severe
drought it may shed most or nearly all of its leaves. Its (white and fragrant) flowers
are arranged axillary, normally in more-or-less drooping panicles. It also bears fruits.
The neem tree is noted for its drought resistance. Neem can grow in many different
types of soil, but it thrives best on well drained deep and sandy soils. It is a typical
tropical to subtropical tree and exists at annual mean temperatures between 21-32 °C.
It can tolerate high to very high temperatures and does not tolerate temperature below
4 °C (142).
The taxonomy of Azadirachta indica is shown below;
Kingdom: Plantae
Division: Magnoliophyta
Order: Rutales
Family: Meliaceae (mahogany family)
Genus: Azadirachta
Species: A. indica
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Numerous pharmacological activities have been ascribed to the various part of the
large evergreen tree, Azadiracta indica. Its leaf extract has been prescribed for oral
use for the treatment of malaria by Indian ayurvedic practitioners from time
immemorial (143). Dried neem leaves in the form of tea are used by the people of
Nigeria and Haiti to treat this disease (144). It is a Nigerian naturalized medicinal
plant known locally as ‘Dogoyaro’, which has been found to have antimalarial
properties in vivo and in vitro (145,146). The antimalarial activity of its leaf extract
has not been found to be great (147). The mechanism of action of neem is believed to
be probably due to redox perturbation in the form of the imposition of substantial
oxidant stress during therapy. The aqueous leaf extract inhibits NADPH-cytochrome c
(P-450) reductase activity in rats with significant increase in microsomal protein
(140). Neem extract have a schizontocidal and gamatocytocidal effect (148,149).
Neem seed and leaf extract are effective against malaria parasites (150,151). The leaf
extract contains; sterols, limonoids, flavonoids and their glycosides and coumarins. Of
interest are gedunin (150, 152, 153), nimbolide (153,154), nimbinin (153), 11-β-
acetoxy gedunin (153), dihydrogedunin (153), meldenin (154), isomeldenin (154),
nimocinol (154), nimbandiol (154), maldenin, azadirachtin and quercetin; which are
responsible for this property. Meldenin was found to be the most active of four
limonoids isolated from its leaves, against the chloroquine- resistant K1 strain of P.
falciparum(155). Azadiractin and three of its semisynthetic derivatives have been
found to inhibit the formation of motile male gametes in vitro (148, 155). This has
raised the possibility of developing an azadiractin-based compound as antimalarial
with transmission-blocking potential (156).
Both alcohol and water extracts of neem leaf have been confirmed as effective. It
blocks the development of the gamete in an infected person. It greatly increases the
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state of oxidation in red blood cells, which prevents the normal development of the
parasite. Some studies show that even chloroquine-resistant strains of malaria are
sensitive to neem, particularly a component called irodin A. 100 % of the malaria
gamete are dead within 72 hrs with a 1:20,000 ration of active ingredients (157).
Gedunine (a limonoid) and quercetin (a flavonoid) compounds found in the leaves are
also effective against malaria. In vitro, gedunin possesses activity about three times
higher than chloroquine, but twenty-times lower than quinine (153). Gedunin has also
been shown to exhibit an additive effect when combined with chloroquine (158,159).
However, despite the promising in vitro activity of gedunin, it has not been found to
inhibit Plasmodium berghei in mice (153). This is probably due to poor
bioavailability, and loss of natural synergy or ability of other constituent to protect it
from digestion and/or enhance its solubility.
Leaves of neem, especially its gedunin content have been shown to be active against
chloroquine resistant strains in vitro and this activity can be used to standardize A.
indica when it is to be used as antimalarial (160). The aqueous extract of neem leaves
has an IC50 of 2µg/ml, with complete inhibition of the schizonts maturation exhibited
at 7.8 µg/ml (161), and shows significant activity at 125-500 mg/kg against P. berghei
(162), and 41.2% parasite suppression was found at this dose range (163). Some
scientists believe that stimulation of the immune system is a major factor in neem’s
effectiveness against malaria. The plant also lowers fever and increase appetite,
enabling a stronger body to fight the parasite and recover more quickly.
The safety evaluation of neem leaf extract reveals that acute and sub-acute effect of
the aqueous leaf extracts are mostly beneficial (164). Intravenously administered
aqueous leaf extract at a dose greater than 40 mg/kg body weight produces toxic
manifestation leading to death in guinea pigs (165). Successive doses of 5–200 mg/kg
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reduce heart rate and increased the arterial pulse rate in guinea pigs (166). Aqueous
leaf extract also shows antifertility effect in mice when given through the oral route
(165,167). Crude neem leaf extracts causes genotoxicity in male mice germ cell at a
dose of 0.5–2 g/kg body weight for 6 weeks. Some structural change in meiotic
chromosomes along with chromosome strand breakage or spindle disturbances and
abnormal regulation of genes controlling sperm shape were observed (168). Neem
leaf extract when administered for 48 days in albino rats causes decrease in sperm
count, sperm motility and forward velocity, probably due to androgen deficiency
(169). Oral administration of 20–60 mg dry leaf powder for 24 days in rats causes
decrease in the weight of seminal vesicle and ventral prostrate and regressive changes
of the histological parameters through its antiandrogenic property (170).
1.8 Methods employed in the evaluation of antimalaria activity of a substance
The antimalarial activity of a substance can be evaluated via the use of the following
models (171);
1.8.1 In vitro methods for screening antimalarial compounds
Different in vitro methods have been developed, viz;
I. 3H-Hypoxanthine uptake method
II. Giemsa stained slide method (MIC method)
III. Use of flow cytometry
IV. Measurement of LDH activity of Plasmodium falciparum
V. Isobologram analysis for combination therapy
1.8.2 In vivo methods for screening antimalarial compounds
Plasmodium species that cause human disease are essentially unable to infect non
primate animal models. So, in vivo evaluation of antimalarial compounds begins with
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the use of rodent malaria parasite. Plasmodium berghei, P. yoelii, P. chabaudi, P.
vinckei have been used extensively in drug discovery and early development. Choice
of rodent malaria species and mouse strains need to be considered during
experimental design and interpretation. P. chabaudi and P. vinckei generate a high
parasitemia and produce synchronous infections (propogation of specific stage),
enabling studies on parasite stage specificity. P. chabaudi and P. vinckei are more
sensitive than P. berghei to iron chelators and lipid biosynthesis inhibitors. In vivo
models include;
I. Rodent models: Here activity is represented as EC (effective concentration).
Such models include;
a. Plasmodium berghei 4 day suppression test
b. Hill’s test for causal prophylaxis and residual activity
c. Sporontocidal activity testing
d. Rane or curative test
e. Use of immunocompromised mice and P. falciparum: Immunocompromised
mice can support Plasmodium falciparum infection as it lacks T and LAK (
Lymphokine activated killer cell ) cells.
II. Avian models, which is no longer popular due to the introduction of rodent
models
III. Primate models: e.g Plasmodium cynomolgi rhesus model
1.8.2.1 Plasmodium berghei 4 day suppression test
This is a preliminary test method when evaluating the antimalaria activity of a
compound. Here, the efficacy of a compound is assessed by comparison of blood
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parasitemia and mouse survival time in treated and untreated mice groups. Naval
Medical Research Institute (NMRI) mice free from Eperythrozoon coccoides and
Haemobartonella muris are the standard of mice to be used and they should be
maintained at 22 ˚C at 50-70 % humidity, fed with diet containing p-aminobenzoic
acid 45 mg/kg and water ad libitum. Mice contaminated with Eperythrozoon
coccoides survive infection with P. berghei longer than clean mice whereas the
presence of Haemobartonella muris tends to accelerate the malaria infection. On day
0, mice are injected with 0.2 ml of aliquot (2X108 parasitized erythrocytes)
intravenously or intraperitoneally. The animals are then grouped into groups of five
mice each. Vehicle treated mice (control group) is compared with the test drug treated
group(s). A positive control group given chloroquine or any other reference drug is
included in the study. The drugs are prepared at required concentration, as a solution
or suspension containing 7 % Tween 80/3 % ethanol and administered 2-4 hr post
infection by appropriate routes. On day 1 to 3, the experimental groups are treated
again (with the same dose and same route) as on day 0. On day 4, 24 h after the last
dose (i.e. 96 h post-infection); thin blood smears from all animals are prepared with
Giemsa stain. Parasitemia is determined microscopically by counting 4 fields of
approximately 100 erythrocytes per field. For low parasitemias (< 1%), up to 4000
erythrocytes have to be counted. The difference between the mean value of the control
group (taken as 100%) and those of the experimental groups is calculated and
expressed as percent reduction or activity using the following equation:
……………………Eqn. 1
For slow acting drugs, additional smears should be taken on days 5 and 6, to
determine parasitemia from which the activity is calculated accordingly. Untreated
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control mice typically die approximately one week after infection. For treated mice
the survival-time (in days) is recorded and the mean survival time is calculated in
comparison with the untreated and standard drug treated groups. Mice without
parasitemia on day 30 of post-infection are considered cured (171).
1.9 Objective of the study
New antimalarial drugs must meet the requirements of rapid efficacy, minimal
toxicity, and low cost. Immediate prospects for drugs to replace chloroquine and SP
include amodiaquine and chlorproguanil-dapsone (another antimalarial acting like
SP). But they already suffer some cross resistance with chloroquine and SP. The
artemisinins are next in line but they have very short half-life necessitating their use in
combination with a long acting drug. This long acting drug should probably be one to
which no known resistance has been developed yet. Moreover, antimalarial
combinations apart from improving efficacy should also forestall loss of antimalarial
activity in the face of resistance to one of the agents in the combination and reduce the
selection of antimalarial drug resistance. Such combinations may also provide for the
reduction of dosages, cost and toxicity. Additional advantages of a combination may
also arise if the individual drugs are active against different stages of the parasite.
Neem is known to be active against asexual forms in the blood and the development
of gametocyte and is also gametocytocidal. Artesunate has gametocytocidal effects.
Thus the combination of two gamecytocidal agent, ie artesunate and neem will further
decrease transmission of malaria plasmodia, even of resistant plasmodia, in high
transmission areas like Nigeria. Pawpaw leaf extract also have antimalarial activity
together with the special effect of preventing the development of anemia. Therefore
its combination with artesunate will improve treatment outcomes and enable patients
to recover quickly.
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This study therefore aims to;
Determine the pharmacodynamic interaction between the combinations of
artesunate and neem or Carica papaya aqueous crude leaf extract, based
on their antimalarial activity.
Provide the basis for the development of an artemisinin-based combination
therapy with crude drugs, so as to provide cost effective ACTs, and thus
bring orthodox and herbal medicines closer together.
Provide the basis for the possible isolation of a particular antimalarial
constituent of the neem or Carica papaya leaf extract, which can be
combined with artesunate, thus making a new and effective orthodox
combination therapy.
CHAPTER TWO
MATERIALS AND METHODS
2.1 MATERIALS
2.1.1 Experimental animals
The mice used in this study were 8-14 weeks old, non-pregnant Swiss albino females
of 25 ± 2 g, obtained from the Faculty of Veterinary Medicine and Faculty of
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Pharmaceutical Sciences, UNN. The mice were left to acclimatize in the experimental
animal house unit of the Department of Biochemistry, UNN, for 5 days, during the
time of which their fed was supplemented with 45 mg/kg p-aminobenzoic acid and
water ad libitum. They were kept in a cool room. The animals were handled according
to the guidelines for laboratory animal use of the University of Nigeria, Nsukka.
2.1.2 The parasite
The parasite used in this study is chloroquine-sensitive Plasmodium berghei, NK 65
strain, obtained from the National Institute of Pharmaceutical Research and
Development (NIPRD), Abuja, and constitute the blood of mice already infected with
P. berghei, serving as the donor mice for further use in this experiment.
2.1.3 Plant extracts
The plants in this study were Azadirachta indica and Carica papaya, which were
identified by Mr. Ozioko of Bioresources Development Centre, BDC, and a voucher
specimen with No. Intercedd (International Centre for ethnomedicine and drug
development) 917 and Intercedd 918 respectively was deposited there. The plant
extract used were aqueous crude extract of the fresh leaves of Azadirachta indica and
mature fresh leaves of Carica papaya.
2.1.4 Chemicals
The pure artesunate powder used in this study was a generous gift from Emzor
Pharmaceutical Industries, Lagos. Other chemicals include; Giemsa stain (Kiran Light
Lab., Mumbai, India), Hayem’s solution, sodium chloride (M&B, England), sodium
dihydrogen phosphate (Kermel, India), Tween 80 (BDH, England), and absolute
methanol and ethanol (Sigma-Aldrich, USA).
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Other materials include; mice cages, 1 ml syringes, heparinized capillary tubes, glass
slides, dissecting set, light microscope, universal indicator paper, distilled water and
improved Neubauer Counting Chamber.
2.2 METHODS
2.2.1 Chemical preparation
2.2.1.1 Extraction of Crude drugs
Fresh leaves of Azadirachta indica (60 g) and Carica papaya (80 g), were separately
cleaned by rinsing in clean water twice, and then were homogenized with 200 ml of
sterile cold distilled water for 24 h. They were then sieved with muslin cloth and the
filtrate stored in the refrigerator. The concentration of the filtrate was then determined
by evaporating a given volume of the supernatant of the filtrate to dryness, and the
concentration in weight/ml was determined. This extraction was done 48 h prior to the
day 0 of the in-vivo schizontocidal activity testing. The extracts were discarded after
storage in the fridge for 7 days, and fresh ones were made as and when needed. The
percentage yield of each crude drug was calculated from;
Yield (%) =
The stock extract of neem was diluted with a mixture of Tween 80 and ethanol in
sterile distilled water, so as to enable the administration of doses of 100, 500 and 1000
mg/kg of the extract, and the concentration of Tween 80 and ethanol was not beyond
7% and 3% respectively. The stock extract of pawpaw was diluted with the same
solvent as for neem, to give doses of 50, 100 and 200 mg/kg. The control and test
groups were given equal volumes of the doses above. The selection of these doses
was based on the effect of neem or pawpaw leaf extract on Plasmodium in infected
mice (162, 139).
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Carica papaya
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Azadirachta indica
Figure 3: Pictures of Carica papaya and Azadirachta indica
2.2.1.2 Preparation of artesunic acid
The artesunic acid powder was dissolved in 7 % Tween 80 and 3 % ethanol in cold
sterile distilled water to give concentrations of 6, 15 and 20 mg/kg. The doses used
were based on the ED50 of artesunate on P. berghei infected mice (172).
2.2.2 In vivo schizontocidal activity of combination of a fixed dose of artesunic
acid with varying doses of crude extracts
The effect of combination therapy on early infection was checked. This was carried
out according to standard protocol following the classical Peter’s 4-day suppressive
test (171).
On day 0 of this test, the percentage parasitemia and red blood cell count of the donor
mice were determined by using a Giemsa-stained thin blood smear of the donor mice
and improved Neubauer Counting Chamber, respectively. The blood of the donor
mice was collected by cardiac puncture and from the retro-orbital plexus vein, and
diluted with physiological saline (normal saline) to give a concentration of 108
parasitized erythrocytes per ml. Parasitized erythrocytes (2 x 107, i.e., 0.2 ml of 10
8
parasitized erythrocytes/ml) was injected intraperitoneally into each of the
experimental mice. The mice was then shared randomly into 10 groups of five mice; a
negative control group (given 7 % Tween 80 and 3 % ethanol in sterile distilled
water), and the test group given the different doses of artesunate and the two crude
extracts. The drug, extracts and placebo were administered orally at 4, 24, 48 and 72 h
post infection. Day 4 (24 h) and day 7 (96 h) after the last treatment, thin blood
smears from the tail vein of all animals were fixed with methanol and stained with 10
% Giemsa solution for 10 min.
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Percentage parasitemia was determined microscopically by counting 4 fields of
approximately 100 erythrocytes per field. The antimalarial activity was calculated by
converting the fractional reduction in parasitemia to percentage as shown in the
equation:
……………Eqn. 1
The mice were left till day 30 post infection during the time of which the mean
survival time of each group was noted, calculated and compared. The mouse that was
still alive on day 30 was checked for parasitemia and those with none were considered
cured. The dose of artesunate which gave a minimally significant reduction in
parasitemia, i.e. 15 mg/kg, was chosen as the fixed dose of artesunate to be combined
with different doses of the crude extract in the next phase of this test.
In the next phase, as explained previously when the test was carried out using drugs
and extracts singly, all groups of animals were given equal volumes of the
combinations of drug and extracts, and placebo. Similarly 24 h and 96 h after
administering the last treatment, blood smears were taken and the percentage
parasitemia determined and used to calculate the antimalarial activity by using the
equation above.
The mice were also left till day 30 post infection during the time of which the mean
survival time of each group was noted, calculated and compared. Each mouse still
alive on day 30 was checked for parasitemia and those with none were considered
cured.
2.2.3 Determination of ED50 of the crude extracts
The dose-response relationship of the crude drugs and artesunic acid was obtained by
plotting the percentage reduction in parasitemia, i.e., antimalarial activity of the drugs
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against the logarithm of their respective doses. The linear equation of the graph was
then used to calculate the ED50 of the drugs in P. berghei infected mice.
2.2.4 Determination of the kind of pharmacodynamic interaction between the
pure drug and plant extracts
Here a dose-response relationship of the crude drugs in combination with artesunate
was obtained by plotting the percentage reduction in parasitemia, i.e., antimalarial
activity of the combination therapy against the logarithm of their respective doses.
The linear equation of the graph was then used to calculate the ED90 of the crude
drugs in combination with artesunic acid in P. berghei infected mice. The ED90 of the
crude drugs alone were also calculated from the equation of their dose-response
relationship already drawn in the previous section. The ED90 was then used to
calculate the isobolar equivalent (IE) of the crude drugs by using the equation (172);
………………………………………….Eqn. 2
However, the IE was not used to draw an isobologram as the Checkerboard technique
was not what was used here.
2.2.5 Data analysis
The significance of treatment effect was evaluated by Games-Howell’s Post Hoc
Multiple Comparism Test instead of Tukey HSD or LSD or Scheffe because the
Levene’s Test for homogeneity of variance showed that the variance of the groups is
significantly different, at p< 0.05.
The significance of survival time was evaluated by Tukey HSD as the Levene’s Test
for homogeneity of variance showed that the variance of the groups are homogeneous
at p<0.05.
CHAPTER THREE
RESULTS AND DISCUSSION
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3.1 Chemical preparation
3.1.1 Percentage yield of crude drugs
The concentration of the extracts varied; 20, 25 and 30 mg/ml for neem and 10, 20
and 35 mg/ml for pawpaw. The percentage yield ranged from 6.67 to 10 % for neem,
and 2.5 to 8.75 % for pawpaw. Therefore, on the average the percentage yield of
neem aqueous crude leaf extract (NCE) was higher (8.33 ± 1.67 %) than that of
pawpaw aqueous crude leaf extract (PCE) (5.42 ± 3.16 %).
3.1.2 Preparation of stock solution of artesunate acid
Artesunate comes in pure powder form, for injection as artesunic acid (173). It is then
dissolved in 5% NaHCO3 and diluted with dextrose water. But, the powder used did
not dissolve in 5 % NaHCO3 but in 5% NaOH. The vehicle (7 % Tween 80 and 3 %
ethanol) used to dissolve artesunic acid (ARTA) here, is specified for antimalarial
work in the literature (171).
3.2 In vivo schizontocidal activity of combination of a fixed dose of artesunate
with varying doses of crude extract.
Appendix II show the pictures of the thin blood smear made during this test, while,
Fig. 4 and 5 show the graphical representation of the mean percentage parasitemia
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values (calculated from the parasitemia values shown in appendix III) of the various
dose levels of each drug and combination, i.e. each treatment, on day 4 and 7.
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The parasitemia level of the untreated group is expected to increase as seen in Fig. 5
for the combination treatment, but that of the single treatment, Fig. 4, did not increase
considerably. This is most probably due to the plasmodiastatic effect that was brought
about by contamination of the donor mice blood by Eperythrozoon coccoides, during
serial passage to maintain the parasite, during passage into test group, or was
transmitted by blood-feeding arthropod vectors like; lice (Polypax spinulosa and
Polypax serrata) (174). Eperythrozoon coccoides is an epierythrocytic organism that
causes mild haemolytic anaemia in laboratory and wild mice and is currently thought
to be a rickettsia. This parasite inhibits the growth of the plasmodium, and enables the
mice to survive longer as seen in Table 3, below even though they are infected with P.
berghei. The parasite can be detected on the erythrocytes by the use of Romanowsky
or acridine orange dyes and a fluoresence microscope, prevented by hygiene and
treated by the use of antibiotics like; organic arsenicals and neoarsphenamine.
Infections produce an intense parasitemia that peaks on day 2 to 5 (acute infection
stage) and subsequently declines rapidly (latent infection stage), so that by day 6 or 7,
the number of organisms in the peripheral blood is very low, and the organism may go
undetected in blood smears unless they are specifically searched for. Death from
infections with Eperythrozoon coccoides is very rare (174).
This may have produced an artificial enhancement in the antimalarial activity, i.e.
ability to suppress parasite growth, of neem 100 mg/kg (66.37%), as shown in Table
1, on day 4. For the single therapy phase, it is assumed that the parasite started
growing from day 6 or 7, when the effect of Eperythrozoon coccoides must have
subsided, thus allowing the P. berghei to multiply. According to a study, when albino
mice harboring a latent infection of Eperythrozoon coccoides are infected with P.
berghei, the former infection remains latent and exerts no influence on the course of
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malaria infection, but if the two infections are introduced concurrently the course of
malaria will be affected (175). From literature, doses of aqueous neem leaf extract
found to be effective are 125 to 500 mg/kg. Here, the percentage reduction is 36.25%
and is not significant at p< 0.05.
When the parasite eventually started growing, on day 7, the parasitemia level
decreased for neem and artesunate in a dose dependent manner, while that of pawpaw
increased in a dose dependent manner, as it has weak activity, Fig.4. Neem and
Pawpaw may also be slow acting. The activity of artesunate decreased in a dose
dependent manner, with artesunate 6 mg/kg losing its activity on day 7, probably due
to recrudescence.
The reduction in the level of parasitemia, compared to the control (5.38%) was
significant for dose level of 1000 mg/kg of neem, 50 mg/kg of pawpaw, and dose
levels of 15 and 20 mg/kg of artesunate(1.56, 1.45,1.6 and 1.5% respectively) on day
7.
The combination treatment depicts clearly the behaviour of P. berghei in both treated
and untreated groups, with the parasitemia levels increasing from day 4 to day 7 for
the untreated and treated groups, Fig.5. The parasitemia of the treated group is
significantly lower for all treated groups on day 4 as compared to the control (4.80
%). On day 7, the parasitemia of the treated group are also significantly lower for all
treated groups as compared to the control (12.25%).
Table 1 shows the antimalarial activity, i.e., the mean percentage reduction in
parasitemia (calculated from the individual reduction in parasitemia shown in
appendix IV) of the drugs alone and in combination, compared to the control.
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Table 1: Effect of artesunic acid and/or NCE or PCE on the growth of P. berghei in
mice, day 4 of checking parasitemia
Treatment Percentage of parasitemia reduction (%)
Arta 6 mg/kg 63.72 ± 16.96*
Arta 15 mg/kg 62.83 ± 17.31*
Arta 20 mg/kg 68.14 ± 8.51*
NCE 100 mg/kg 66.37 ± 11.54*
NCE 500 mg/kg 36.25 ± 32.07
NCE 1000 mg/kg 40.71 ± 23.54
PCE 50 mg/kg 37.70 ± 11.42
PCE 100 mg/kg 34.73 ± 24.34
PCE 200 mg/kg 59.29 ± 20.37*
Arta 15 mg/kg + NCE 100 mg/kg 79.17 ± 20 .50*
Arta 15 mg/kg + NCE 500 mg/kg 83.33 ± 11.88**
Arta 15 mg/kg + NCE 1000 mg/kg 96.87 ± 2.85***
Arta 15 mg/kg + PCE 50 mg/kg 81.25 ± 5.94****
Arta 15 mg/kg + PCE 100 mg/kg 76.04 ± 17.89*
Arta 15 mg/kg + PCE 200 mg/kg 58.04 ± 12.05*
*- Significantly greater than the control, **- significantly greater than the control &
pawpaw 50 mg,***-significantly greater than the control, pawpaw 50 mg, art 20 mg
and arta 15 mg + pawpaw 50 mg/ pawpaw 200 mg, ****- significantly greater than
pawpaw 50mg all at p< 0.05
Table 2 : Effect of artesunic acid and/or NCE or PCE on the growth of P. berghei in
mice, day 7 of checking parasitemia
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Treatment Percentage reduction in parasitemia (%)
Arta 6 mg/kg 37.27± 18.78
Arta 15 mg/kg 70.26± 11.66*
Arta 20 mg/kg 72.12± 13.55*
NCE 100 mg/kg 37.27± 21.63
NCE 500 mg/kg 52.60± 21.40
NCE 1000 mg/kg 72.12± 13.15*
PCE 50 mg/kg 73.05± 10.07*
PCE 100 mg/kg 47.03± 19.33
PCE 200 mg/kg 48.96± 12.73
Arta 15 mg/kg + NCE 100 mg/kg 77.30± 14.70*
Arta 15 mg/kg + NCE 500 mg/kg 84.08± 5.67*
Arta 15 mg/kg + NCE 1000 mg/kg 89.80± 4.02*
Arta 15 mg/kg + PCE 50 mg/kg 57.59± 16.69*
Arta 15 mg/kg + PCE 100 mg/kg 72.57± 17.11*
Arta 15 mg/kg + PCE 200 mg/kg 81.40± 6.66*
*- Significantly greater than the control at p< 0.05
Table 1, shows that the treatment of P. berghei infected mice with 6 mg/kg of
artesunate, produced a significant reduction in parasitemia (63.72%) compared to the
control on day 4. Higher doses of artesunate 15 and 20 mg/kg equally significantly
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suppressed the parasitemia of the infected mice to give activities of 62.83 and 68.14
% respectively.
P. berghei infected mice treated with 100 mg/kg of neem and 200 mg/kg of pawpaw
experienced a significant reduction in their parasitemia levels (66.37 and 59.29 %
respectively), while those treated with 500 and 1000 mg/kg of neem, and 50 and 100
mg/kg of pawpaw experienced a mild reduction in their parasitemia levels compared
to the untreated group on day 4.
On day 7, the reduction in parasitemia of the single treatment group decreased for the
treatment groups that previously showed a significant reduction in parasitemia,
(artesunic acid 6mg/kg, pawpaw 200 mg/kg and neem 100 mg/kg), probably due to
recrudescence as a result of Eperythrozoon coccoides plasmodiastatic effect. But,
artesunic acid 15 and 20 mg/kg still retained their activity.
Paradoxically, pawpaw 50 mg/kg, significantly suppressed the parasitemia of the
infected mice on day 7 (69.09%), compared to the control. This may be due to its
slow activity. Increasing the dose of pawpaw produced a mild reduction in
parasitemia on day 7, probably due to ingredients of the complex mixture of the
pawpaw crude extract antagonizing the activity of one another at higher dose levels.
The combination treatment yielded a more significant reduction in parasitemia
compared to the control on day 4. The combination of artesunic acid 15 mg/kg and
pawpaw 50 mg/kg, produced a significant reduction in parasitemia compared to
pawpaw50mg/kg, alone, Table 1. The artesunate thus, enhanced the antimalarial
activity of pawpaw via a pharmacodynamic interaction.
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The combination of artesunate 15 mg/kg and neem 1000 mg/kg, produced a
significant reduction in parasitemia than artesunate 20 mg/kg, alone, the combination
of artesunic acid 15 mg/kg and pawpaw 50 mg/kg, pawpaw 50 mg/kg and the
combination of artesunic acid 15 mg/kg and pawpaw 200 mg/kg, Table 1. Even on
day 7, activity of this combination still remained significantly high (89.80 %), Table
2, although, it was not significantly higher than artesunic acid, 20mg/kg, alone. Neem
thus, enhances the antimalarial activity of artesunate via a pharmacodynamic
interaction and a pharmacokinetic interaction, as neem has been shown to increase the
serum concentrations of artesunate in our laboratory (176).
The combination of artesunic acid 15 mg/kg and neem 500 mg/kg produced a
significant reduction in parasitemia compared to pawpaw 50 mg/kg alone.
On day 7, all the combinations of artesunic acid 15 mg/kg and the crude extract still
retained their antimalarial activities.
3.3 Survival time and percentage cure of P. beghei infected mice after treatment
Table 3 and 4, show the survival time based on a 30 days observation period,
percentage survival at the end of this period and percentage of the infected mice that
are cured, i.e. do not have any parasite in their blood at day 30.
Table 3: Effect of single treatment (PCE or NCE or artesunic acid) to P. berghei
infected mice survival and percentage cure
Experimental
condition
Mean survival time
(days)
Percentage survival
(%)
Percentage cure
(%)
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Control Group 19.80 ± 4.44 0.00 0.00
Arta 6 mg/kg 21.00 ± 3.74 0.00 0.00
Arta 15 mg/kg 21.80 ± 3.34* 0.00 0.00
Arta 20 mg/kg 22.80 ± 2.28* 0.00 0.00
NCE 100 mg/kg 11.20 ± 8.29 0.00 0.00
NCE 500 mg/kg 23.80 ± 4.76* 20.00 0.00
NCE 1000 mg/kg 18.20 ± 4.32 0.00 0.00
PCE 50 mg/kg 21.00 ± 3.74 0.00 0.00
PCE 100 mg/kg 17.40 ± 4.93 0.00 0.00
PCE 200 mg/kg 17.60 ± 5.27 0.00 0.00
* - Significant when compared to NCE 100 mg/kg at p< 0.05
Table 4: Effect of combination treatment (PCE or NCE and artesunic acid ) to P.
berghei infected mice survival and percentage cure
Experimental
condition
Mean survival time
(days)
Percentage
survival (%)
Percentage cure
(%)
Control group 11.20±4.55 0.00 0.00
Arta 15 mg + NCE 100 28.20±4.02* 80.00 40.00
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mg/kg
Arta 15 mg + NCE 500
mg/kg
28.20±4.02* 80.00 60.00
Arta 15 mg + NCE
1000 mg/kg
28.20±4.02* 80.00 40.00
Arta 15 mg + PCE 50
mg/kg
23.00±6.60* 40.00 20.00
Arta 15 mg + PCE 100
mg/kg
22.60±8.62* 40.00 20.00
Arta 15 mg + PCE 200
mg/kg
19.60±5.94 20.00 20.00
* - Significant when compared to control at p< 0.05
The group treated with neem 100 mg/kg survived for an average of 11.20 days, its
survival time being lower than that of control group (19.8 days), and significantly
lower than that of the groups given neem 500 mg/kg, artesunate 15 and 20 mg/kg. Its
percentage survival and cure rate are 0%.
The survival times of the following groups are equally lower than that of the control
group, but not significantly lower; neem 1000 mg/kg, pawpaw 100 and 200 mg/kg.
This may be due to their toxicity at this dose level. This also, contributes to the
paradoxical effect of pawpaw 50 mg/kg, as it would have been expected to have a
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lower survival time as higher doses of the crude extract did. Their percentage survival
and cure rate are equally 0%. It is only one mouse in the group that received 500
mg/kg of neem that survived up till day 30, even so the mouse was still infected, thus
making their percentage survival to be 20% and their cure rate to be 0%.
For the combination treatment, all the treatment groups survived significantly
compared to the control (11.20 days, 0%), except for the combination of artesunate 15
mg/kg and papaw 200 mg/kg as shown in table 4. This further justifies its least
percentage in reduction in parasitemia, Table 2.
At least one of the mice in all the combination treatment group were cured, with the
highest cure rate being 60 % (for the combination of artesunate 15 mg and neem 500
mg) and the lowest cure rate being 20 % for all the combinations of artesunate and
pawpaw.
The percentage survival is highest for all the combinations of artesunate and neem
(80%), and lowest for the combination of artesunate 15 mg/kg and pawpaw 200
mg/kg (20 %). Possibly, some constituents of this high dose of the crude extract of
pawpaw are high enough to antagonize to an extent the antimalarial activity of some
other constituent of pawpaw leaf or artesunate itself. Pawpaw is known to have
antioxidant effect, it being a free radical scavenger, and helping with splenomegaly,
while artesunate is known to act by being converted to a free radical. Thus, a
pharmacological antagonism may be occurring at some minute level, thus inhibiting
to an extent the activity of artesunate. The toxicity of pawpaw at this dose may also
play a role in the reduction of the survival time, percentage survival and cure rate of
this combination.
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The percentage survival and cure rates are higher for all combinations than the extract
or pure drug given alone, except for the combination of artesunate 15 mg/kg and
pawpaw 200 mg/kg, whose percentage survival is equal to that of neem 500 mg/kg
group. Its survival time is equal to that of the control in the single treatment group.
The survival time for the combination of artesunate 15 mg/kg and pawpaw 50 mg/kg
is equal to that of neem 500 mg/kg.
3.4 Determination of ED50 of the crude extracts
Due to the plasmodiastatic effect brought about by Eperthrozoon coccoides, the ED50
of artesunic acid, and aqueous extract of neem and pawpaw leaf was calculated based
on day 7 not day 4.The ED50 of the drugs was calculated by using the linear equation
of the dose-response relationship of the drugs, shown in Fig. 6,7 and 8.
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The ED50 of artesunate, neem and pawpaw aqueous leaf extract were; 8.814, 277.95
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and 143.53 mg/kg respectively. The ED50 of artesunic acid agrees with the ED50 of
artesunate against P. berghei, ANKA strain from literature, and is 8mg/kg (177).
The in vivo anti-plasmodial activity of the crude extract can be classified as moderate,
good and very good depending on if the extract displays a percentage growth
inhibition equal to or greater than 50% at a dose of 500, 250 and 100 mg/kg
respectively (178). Based on this, NCE has a moderate activity, while PCE has a very
good activity.
3.5 Determination of the kind of pharmacodynamic interaction between the pure
drug and plant extracts.
The ED90 of the drug alone and in combination was calculated from the linear
equation of the dose-response relationship of the drugs alone (on day 7) and in
combination (on day 7). (Fig. 6-10)
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The ED90 was then used to calculate the isobolar equivalent (IE) of NCE and PCE.
The IEs of NCE and PCE were 0.26 and 22.29 respectively. The IE of artesunic acid
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could not be calculated as its dose was fixed in the combination. Based on the IE, the
kind of pharmacodynamic interaction between the crude extracts and artesunic acid
was determined from the criteria; synergistic effect (IE < 1), additive effect (IE = 1),
antagonistic effect (IE > 1) (173).
Based on this, combinations of artesunate and NCE are synergistic for neem, while
the combinations of artesunate and PCE are antagonistic for pawpaw.
CHAPTER FOUR
CONCLUSIONS AND RECOMMENDATION
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Most antimalarial drugs that are now in use were not developed on the basis of
rationally identified targets, but following serendipitous identification of the
antimalarial activity of natural products, for example; quinine and artemisinin,
compounds chemically related to natural products, for example chloroquine and
artesunate, or compounds active against other infectious agents, for example; the
antifolates and tetracycline. With the notable exception of the artemisinin family of
drugs, almost all antimalarial drugs developed till date are active against asexual stage
of the parasite and therefore do not prevent transmission of malaria. Transmission
blocking activity is a desirable property for a new antimalarial drug.
This study combines two antimalarial drugs with transmission blocking activity; neem
and artesunate and then pawpaw and artesunate. Neem enhanced the antimalarial
activity of artesunate, while artesunate enhanced the antimalarial activity of pawpaw.
The combinations of artesunate and neem also prolonged the survival of the treated
infected mice, compared to artesunate alone. This combinations even produced a cure
(at least 40%), while the doses of artesunate used in this study did not produce any
cure at the end of the 30 day period of this study. The combinations of artesunate and
pawpaw also prolonged the survival time of the infected mice and increased the cure
rate, compared to artesunate alone, but the percentage survival and cure rate was
lower than that of the combinations of artesunate and neem aqueous crude extract.
However, the combinations of artesunate and pawpaw are antagonistic for pawpaw,
despite the fact that artesunate enhances its activity, because its IE is 22.29, and even
its percentage survival is lowest for the combination of its highest dose with
artesunate. Combinations of artesunate and pawpaw show little promise.
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The combinations of neem and artesunate are synergistic for neem. Therefore, this
combination is therefore a promising candidate for a new antimalarial combination
therapy development, although the dose level of the neem extract is high. The
findings in this study show that it is possible to have a neem constituent- based
combination therapy, and this is supported by an earlier finding that Azadirachtin-
based compounds as antimalarial agents(with transmission blocking potential) can be
developed as Azadirachtin of A. indica was able to block the development of motile
malaria gametes in vitro.
Other techniques can be used to evaluate the drug interaction between artesunate and
neem leaf extract, so as to determine if such combinations are synergistic, additive or
antagonistic for artesunate.
Isolation of a particular antimalarial constituent of the neem leaf extract, which can be
combined with artesunate, can be done, thus making a new and effective orthodox
combination therapy.
The neem aqueous leaf extract may be toxic to infants and pregnant women at the
dose levels used in the combinations, and so the combinations may also be toxic to
them.
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APPENDICES
Appendix I: Percentage yield obtained after extraction.
Plant Extract Yield 1(%) Yield 2(%) Yield 3(%)
Neem aqueous crude leaf 6.67 8.33 10
Pawpaw aqueous crude leaf 2.50 5.00 8.75
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Appendix II: Pictures of thin blood smear made for determination of the in vivo
schizontocidal activity of the drugs alone and in combination.
Day 0 Day 30 not cured
Day 4 Day 30 cured
Ring form
of P.
berghei
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Appendix III: Parasitemia level of each mouse used in the evaluation of the in vivo
schizontocidal activity of drugs alone and in combination.
Experimental condition
Parasitemia/mice (%)
1 2 3 4 5
Day 4 of drugs alone
Control 6.75 4.75 4.75 5.00 7.00
Neem 100 mg/kg 1.00 1.75 2.75 1.75 2.25
Neem 500 mg/kg NA 5.00 4.25 NA 1.56
Neem 1000 mg/kg 3.75 4.00 5.00 2.25 1.75
Pawpaw 50 mg/kg 3.00 3.75 NA 4.33 3.00
Pawpaw 100mg/kg 5.75 3.00 3.00 3.00 NA
Pawpaw 200 mg/kg 1.75 4.25 2.00 2.25 1.25
Artesunate 6 mg/kg 2.00 2.75 3.25 1.00 1.25
Artesunate 15 mg/kg 1.5 1.75 3.25 1.00 3.00
Artesunate 20 mg/kg 1.25 2.00 1.75 1.50 2.50
Day 7 drugs alone
Control 6.50 4.25 6.50 4.25 NA
Neem 100 mg/kg 4.50 4.25 2.50 2.25 NA
Neem 500 mg/kg 4.00 1.25 2.00 2.00 3.50
Neem 1000 mg/kg 1.25 1.25 1.00 2.75 1.25
Pawpaw 50 mg/kg 2.25 1.00 1.00 1.75 1.25
Pawpaw 100 mg/kg 3.00 2.25 3.25 1.50 4.25
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Pawpaw 200 mg/kg 3.25 3.00 3.00 1.75 NA
Artesunate 6 mg/kg 2.75 4.75 2.50 3.50 NA
Artesunate 15 mg/kg 1.00 1.00 1.75 2.50 1.75
Artesunate 20 mg/kg 1.50 1.25 2.75 1.00 1.00
Day 4 drugs in combination
Control 3.75 5.25 1.25 6.00 7.75
Art 15 mg/kg + Neem 100 mg/kg 0.50 0.50 0.50 2.75 0.75
Art 15 mg/kg + Neem 500 mg/kg 1.75 0.25 0.75 0.50 0.75
Art 15 mg/kg + Neem 1000 mg/kg 0.00 0.25 0.00 0.25 0.25
Art 15 mg/kg + Pawpaw 50 mg/kg 1.00 1.00 1.25 0.75 0.50
Art 15 mg/kg + Pawpaw 100 mg/kg 2.50 0.50 0.50 1.50 0.75
Art 15 mg/kg + Pawpaw 200 mg/kg 2.75 1.71 2.36 1.25 NA
Day 7 drugs in combination
Control 7.00 7.00 22.75 NA NA
Art 15 mg/kg + Neem 100 mg/kg 2.00 2.03 2.00 1.88 6.00
Art 15 mg/kg + Neem 500 mg/kg 2.25 2.25 1.50 1.00 2.75
Art 15 mg/kg + Neem 1000 mg/kg 1.25 0.50 1.88 1.38 1.25
Art 15 mg/kg + Pawpaw 50 mg/kg 8.00 4.00 6.00 5.38 2.60
Art 15 mg/kg + Pawpaw 100 mg/kg 2.00 3.03 7.00 2.87 1.90
Art 15 mg/kg + Pawpaw 200 mg/kg 3.08 1.13 2.81 2.63 1.75
Appendix IV: Percentage reduction in parasitemia of drugs alone and in combination
Experimental condition Reduction in parasitemia (%)
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1 2 3 4 5
Day 4 of drugs alone
Control 0.00 0.00 0.00 0.00 0.00
Neem 100 mg/kg 82.30 69.03 51.33 69.03 60.08
Neem 500 mg/kg NA 11.50 24.78 NA 72.48
Neem 1000 mg/kg 33.63 29.20 11.50 60.18 69.03
Pawpaw 50 mg/kg 46.90 33.63 NA 23.36 46.90
Pawpaw 100mg/kg -1.77 46.90 46.90 46.90 NA
Pawpaw 200 mg/kg 69.03 24.78 64.60 60.18 77.88
Artesunate 6 mg/kg 64.60 51.33 42.48 82.30 77.88
Artesunate 15 mg/kg 73.45 69.03 42.48 82.30 46.90
Artesunate 20 mg/kg 77.88 64.60 69.03 73.45 55.75
Day 7 drugs alone
Control 0.00 0.00 0.00 0.00 0.00
Neem 100 mg/kg 16.36 21.00 53.53 58.18 NA
Neem 500 mg/kg 25.65 76.77 62.83 62.83 34.14
Neem 1000 mg/kg 76.77 76.77 81.41 48.88 76.77
Pawpaw 50 mg/kg 58.18 81.41 81.41 67.47 76.77
Pawpaw 100 mg/kg 44.24 58.18 39.59 72.12 21.00
Pawpaw 200 mg/kg 39.59 44.24 44.24 67.47 NA
Artesunate 6 mg/kg 48.88 11.71 53.53 34.94 NA
Artesunate 15 mg/kg 81.41 81.41 67.47 53.53 67.47
Artesunate 20 mg/kg 72.12 76.77 48.88 81.41 81.41
Day 4 drugs in combination
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Control 0.00 0.00 0.00 0.00 0.00
Art 15 mg/kg + Neem 100 mg/kg 89.58 89.58 89.58 42.71 84.38
Art 15 mg/kg + Neem 500 mg/kg 63.54 94.79 84.38 89.58 84.38
Art 15 mg/kg + Neem 1000 mg/kg 100.00 94.79 100.00 94.79 94.79
Art 15 mg/kg + Pawpaw 50 mg/kg 79.17 79.17 73.96 84.38 89.58
Art 15 mg/kg + Pawpaw 100 mg/kg 47.92 89.58 89.58 68.75 84.38
Art 15 mg/kg + Pawpaw 200 mg/kg 42.71 64.38 50.83 73.96 NA
Day 7 drugs in combination
Control 0.00 0.00 0.00 NA NA
Art 15 mg/kg + Neem 100 mg/kg 83.67 83.45 83.67 84.69 51.02
Art 15 mg/kg + Neem 500 mg/kg 81.63 81.63 87.76 91.84 77.55
Art 15 mg/kg + Neem 1000 mg/kg 89.80 95.92 84.69 88.78 89.80
Art 15 mg/kg + Pawpaw 50 mg/kg 34.69 67.35 51.02 56.12 78.78
Art 15 mg/kg + Pawpaw 100 mg/kg 83.67 75.26 42.86 76.57 84.49
Art 15 mg/kg + Pawpaw 200 mg/kg 74.86 90.82 77.06 78.53 85.71
Appendix 5: Survival Time in days for each group
Experimental condition
Survival time (days)
1 2 3 4 5
Drugs alone
Control 14.00 17.00 20.00 23.00 25.00
Neem 100 mg/kg 6.00 6.00 9.00 12.00 25.00
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Neem 500 mg/kg 19.00 19.00 25.00 26.00 30.00
Neem 1000 mg/kg 12.00 16.00 19.00 21.00 23.00
Pawpaw 50 mg/kg 17.00 17.00 23.00 23.00 25.00
Pawpaw 100mg/kg 9.00 17.00 20.00 20.00 21.00
Pawpaw 200 mg/kg 9.00 17.00 19.00 20.00 23.00
Artesunate 6 mg/kg 17.00 19.00 19.00 25.00 25.00
Artesunate 15 mg/kg 19.00 19.00 21.00 23.00 27.00
Artesunate 20 mg/kg 20.00 21.00 23.00 25.00 25.00
Drugs in combination
Control 6.00 9.00 9.00 16.00 16.00
Art 15 mg/kg + Neem 100 mg/kg 21.00 30.00 30.00 30.00 30.00
Art 15 mg/kg + Neem 500 mg/kg 21.00 30.00 30.00 30.00 30.00
Art 15 mg/kg + Neem 1000 mg/kg 21.00 30.00 30.00 30.00 30.00
Art 15 mg/kg + Pawpaw 50 mg/kg 17.00 17.00 21.00 30.00 30.00
Art 15 mg/kg + Pawpaw 100 mg/kg 9.00 21.00 23.00 30.00 30.00
Art 15 mg/kg + Pawpaw 200 mg/kg 16.00 16.00 17.00 19.00 30.00
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