Mutations in pfmdr1 Modulate the sensitivity of Plasmodium falciparum to the intrinsic...

Mahidol University International CollegeB.S. (Biomedical Sciences)/ 1

CHAPTER I

INTRODUCTION

1.1 Background

Plasmodium falciparum malaria is increasingly difficult

to treat and control due to the emergence of parasite

resistance to the major antimalarial drug, conspicuously

chloroquine as mentioned above. Early detection of

failing treatment regimens for malaria is important for

guiding public health measures in areas where the disease

is endemic. Thus, such decision making relies on results

of clinical studies that assess the therapeutic efficacy

of antimalarials, sometimes support by in vitro

sensitivity testing.

Currently, molecular genotyping of parasites have

proved advantage in assessing resistance to the

antifolate, sulphonamide, and hydroxynaphthaquinone

classes of drugs, since point mutations in the genes that

encode their drug targets cause resistance (1).

Chloroquine resistance in vitro and in vivo is associated

with mutations in pfmdr1 (Plasmodium falciparum multidrug

resistance gene 1), a putative transporter that modulates

intraparasitic drug concentrations.

Pattaraporn Kanyamee Introduction/ 2

Human falciparum malaria remains a serious disease in

much of the tropical and subtropical world. There are at

least 300 million new infections every single year causing

an estimated 2 million deaths mostly of young children.

Due to its specificity, stability, and safety, chloroquine

has been one of the most successful and widely used

antimalarial drugs (2). The biological activity of

chloroquine is directed against the intraerythrocytic

stage of Plasmodium infection. However, the evolution and

geographical spread of Plasmodium falciparum trophozoites

resistant to chloroquine has greatly reduced the clinical

effectiveness of this compound (3). Identification of

biochemical mechanism responsible for chloroquine

resistance would therefore assist in the development of

alternative chemotherapeutic strategies. The heme

polymerizing activity contained in extracts of both

chloroquine- resistant and chloroquine- sensitive

trophozoites has similar sensitivity to inhibition by

chloroquine (18). Therefore, the mechanism of resistance

must involve either the differential sequestration or

uptake and transport of chloroquine within the parasites.


Figure 1: Malaria distribution and reported drug

resistance (WHO, 1996).

For decades, the treatment of malaria has largely

depended on the use of chloroquine (CQ), a 4-

aminoquinoline recognized for its rapid efficacy, low

toxicity, widespread availability, and affordability. The

emergence and spread of CQ-resistant strains of P. falciparum

has been identified as a major factor responsible for the

recent increases in malaria mortality and morbidity.

Hence, this study is aim to determine the innate

antiplasmodial activity and sensitivity of verapamil on in

vitro chloroquine resistance P. falciparum. Since verapamil

is a weak base which, in addition to acting as a reverser

of chloroquine resistance in the P. falciparum, has itself an


intrinsic antiplasmodial activity. The activity is

dependent of its chloroquine resistance reversal effect,

as the susceptibility of chloroquine-sensitive parasites

to chloroquine is unaltered even in the presence of highly

toxic concentration of verapamil, where as verapamil

alters the susceptibility of chloroquine resistance

parasites to chloroquine at both toxic and non-toxic

concentration.

Drug resistance

Antimalarial drug resistance is the availability of a

parasite strain to survive or multiply despite the

administration and absorption of a drug given in doses

equal to or higher than dose usually recommended, but

within the limits of tolerance of the subject.

Resistance to antimalarial drugs arises as a result

of spontaneously-occurring mutations that affect the

structure and activity at the molecular level of drug

target in the malaria parasite that affect the access of

the drug to that target. The evolution of drug resistance

in Plasmodium is not fully understood although the

molecular basis for resistance is becoming clearer.

Various factors relating to drug, parasite and human host

interactions contribute to the development and spread of

drug resistance. The molecular mechanism of drug action,

drug with a long terminal elimination half-life enhance


the development of resistance, particularly in the area of

high transmission.

1.2 Objectives

The objectives of this study were as follows:

1.2.1 To determine the innate antiplasmodial activity

and sensitivity of verapamil in Plasmodium falciparum

isolates

1.3 Scope of Study

The scopes of this study were as follows:

1.3.1 Twenty Plasmodium falciparum strains which are

parasite isolates: BCI, BC5, BC6, BC11, BC13, BC24,

BC28, PCM4, PCM14, PCM8, TM5, TM6, RN3, T994, J13,

M12, K14C, G112, BC21 and BC35 were taken from many

sources both are wild type and mutation.

1.3.2 Each strain was definitely divided to operate in

the different concentration of verapamil in order to

find the average IC 50 and find the suitable fixing

value of each strain.

1.3.3 Each experiment of different verapamil

concentration was done twice a time per one strain.

1.3.4 The whole experiment was performed in Department

of Parasitology, Pramongkutklao College of Medicine


CHAPTER II


LITERATURE REVIEW

1. Malaria and Plasmodium falciparum

Malaria is a serious, acute and chronic relapsing

infection to humans. It is characterized by periodic

attacks of chills, fever, nausea, vomiting, back pain,

increased sweating anemia, splenomegaly (enlargement of

the spleen) and often-fatal complications. Malaria is also

found in apes, monkeys, rats, birds and reptiles (1, 2).

An infection of malaria in human is caused by one or more

of four species of protozoa parasite, (one-cell organisms)

called sporozoans (subphylum Sporozoa) belonging to the

genus Plasmodium namely P. falciparum, P. vivax, P. malariae and P.

ovale. These parasites are transmitted to human by the bite

of female mosquito belonging to the genus Anopheles, which

has about 60 different species (2). Anopheline mosquitoes

are the only known vectors of malaria in human that

perform this function throughout the world. These

mosquitoes undergo an aquatic larval stage, pupate and

then hatch into flying adults. The females require a meal

of blood to produce fertile eggs and females of some

species prefer human to animal blood. The female mosquito

ingests the malarial parasite by biting a human who was

already infected with the parasite.


Figure 2.1: Anopheline mosquito, the carrier of Plasmodium

parasite.

Life cycle


Figure 2.2: Life cycle of Plasmodium parasites

The malarial parasite has a complicated double life

cycle: a sexual reproductive cycle while it lives in the

mosquito and an asexual reproductive cycle while in the

human host. While it was in its asexual, free-swimming

stage, when it is known as a sporozoite, the malarial

parasite is injected into the human bloodstream by a

mosquito; passing through the skin along with the latter's

saliva. The sporozoite eventually enters a red blood cell

of its human host, where it goes through ring-shaped and


amoeba-like forms before fissioning (dividing) into

smaller forms called merozoites. The red blood cell

containing these merozoites then ruptures and releases

them into the bloodstream (and also causes the chills and

fever that are typical symptoms of the disease). The

merozoites can then infect other red blood cells and their

cycles of development are repeated.

Figure 2.3: Trophozoite and ring phase of Plasmodium

falciparum

Source:

http://www.micro.msb.le.ac.uk/224/malaria.html

A small proportion of the merozoites, however,

becomes gametocytes or germ cells and can go through a

sexual reproductive cycle once back in a mosquito. After a

mosquito has bitten an infected human host and has


ingested the gametocytes, the separate male and female

gametocytes pair off in the mosquito's stomach and they

unite to form a single-celled zygote, which will become an

oocyst. This oocyst eventually divides and releases a

multitude of (asexual, free-swimming) sporozoites that

migrate to the mosquito's head and salivary glands, where

they are ready to pass into the human bloodstream during

the mosquito's next bite. The entire (asexual) cycle is

then repeated (3).

A remarkable feature of the asexual cycle is

that the parasites grow and divide synchronously and the

resulting mass fissions (into merozoites) produce the

regularly recurring attacks or paroxysms, which are

typical of malaria. A malarial attack normally lasts 4 to

10 hours and consists successively of a stage of shaking

and chills; a stage of fever, with the temperature

reaching 105 and severe headache. A stage of profuse

sweat during which the temperature drops back to normal.

Between attacks, the temperature may be normal or below

normal. In the early days of the infection, the attacks

may occur every day, but they soon begin appearing at

regular intervals of either 48 hours (called tertian

malaria) or 72 hours (called quartan malaria). The first

attack usually occurs from 8 to 25 days after an infected

mosquito has bitten a person (3).


The worst type is caused by Plasmodium falciparum.

Complications of P. falciparum malaria include cerebral

malaria, in which the brain infected, severe malaria, in

which the parasitic infection essentially “run out of

control” and placental malaria, in which P. falciparum is

a grave complication of pregnancy, and coma (5). Each of

these complications is very serious and often fatal.

Malaria occurs throughout the tropical and

subtropical regions of the world and is the most prevalent

of all serious infectious diseases. The World Health

Organization estimates that there are over one million

child deaths per year in sub-Saharan Africa and there are

300-500 million cases of malaria per year. More than two

billion people or total 41% of the world’s population

throughout the world (e.g., part of Africa, Asia, the

Middle East, Central and South America, Hispania and

Oceania) live in areas where malaria is transmitted

regularly and there are approximately 1.5-2.7 million

people who die from malaria each year (4).

The incidences of malaria have increased in many

regions in the world and in areas where people thought

that they were diseases free (6). One of malaria factors

that lead to the malaria transmission is travelers or

movement of population. The spread of disease is enhanced

when population move from that place to the others. Most

of malaria cases are imported from regions of the world


where malaria transmission is known to occur (7). This

causes the parasitic resistant to antimalarial drugs.

At present, in Thailand, malaria situation in Thailand

is quite dire; malaria is still an important problem,

especially for malaria control programs (8). The incidence

of malaria morbidity throughout the country has been

increasing during the period 1979-1981. This is attributed

mainly to population movements and problems of parasite

resistance to drugs. The problem of multi-drug resistance

that is associated with population movement is found both in

Thai- Cambodia border and Thai- Myanmar border (9). The

first cases of malaria resistance were found however, along

the Thai-Cambodia border. It is believed that the area of

strongest drug resistance is in the Thai-Kampuchean border

provinces and in the areas adjacent to this border. One of

three major problems confronting the Anti-Malaria Program in

Thailand is the occupational migration of those people for

especially gem mining and farm labor as well as local

migration from villages to forests for woodcutting and

expanding cultivation in foothill forest fringe areas.

Moreover, Pinichpongse (1) mentioned that drug resistance

must be taken into account in the control of malaria among

the migrant population.

2. The Treatment of Malaria: The Basic Chemotherapy


The treatment of malaria aims at eradication or

control of parasite and dealing with clinical

complications. The clinical effects of malaria produce

from the presence of the schizont phase of the parasites

in the erythrocytes. Control of attack depends on the

removal of these parasites from the blood and on the anti-

inflammatory activity of drug used. Drugs which destroy

the schizont forms are called schizonticides. The

classical compounds are the 4- amino- quinolines,

mepracrine, proguanil and primethamine (8).

In the other infections the destruction of any

persistent liver infection is achieved by only one group

of drugs, the 8- amino- quinolines for example, Primaquine

(9).

The following drugs are the important available

compounds used in Southeast Asia and Africa (9, 10, 11,

and 12).

2.1. Chloroquine, Nevaquine and other 4- amino-

quinolines are bitter colorless drug. Parasite

resistance to 4- amino- quinolines, with cross-

resistance to mepacrine, has been demonstrated

in Southeast Asia and Africa (11).


Figure 2.4: The demonstration of chemical

structure of chloroquine.

2.2. Quinine: a bitter crystallines alkaloid

prepared as bihydrochloride, hydrochloride and

bi sulphate. This drug is widely use for long

time since it is easy to find and the resistance

is not much yet.

2.3. Proguanil (Paludrine: Chloroguanide): a

colorless bitter synthetic biguanide. Parasite

resistance is widespread but localized and

erratic in its distribution and degree. Both of

these compounds inhibit the action of

dihydrofolate reductase.

2.4. Pyrimethamine (Daraprim): A colorless

relatively tasteless drug, widely used as a

suppressant, it produces radical cure in

established P. falciparum infection but not other


infections. Parasite resistance is also

widespread. Cross- resistance to proguanil has

been reported.

2.5. Primaquine: bitter colorless synthetic 8-

amino-quinolines: The 8-amino-quinolines are

relatively weak schizonticides but have

considerable activity against the pre-

erythrocytic phase of P. falciparum but only in

toxic dose, meanwhile they actively destroy

gametocytes of all species (11).

2.6 Mepacrine (Artbrin, Atabrine, and Quinacrine):

a bitter yellow acridine compound prepared as the

hydrochloride or methane sulphonate now seldom

used.

2.7 Sulfadoxine: a long-acting sulphamide has

considerable schizonticidal activity and is given

(usually in combination with pyrimethamine as

‘Fansider’) for chemotherapy and chemo suppression

of Chloroquine resistant and proguanil/

pyrimethamine- resistant- plasmodium falciparum

strains

2.8 Diaphynylsulphone (Dapsone, DDS): widely used

in leprosy, has schizonticidal activity against

Plasmodium falciparum in semi- immunes and is also


active against Chloroquine- resistant strains

(12).

2.9 Mefloquine is a trifluoromethyl-

4quinolinemethanol, with a structure similar to

that of quinine. It is a powerful schizonticide,

acting on plasmodium falciparum resistant to

chloroquine, sulfonamide and pyrimethamine. It

has no effect on the liver forms of parasites or

gametocytes (13).

Figure 2.5: The demonstration of chemical

structure of mefloquine.

2.10 Artemisinine (or Qinghaosu): was found to be

a form of sesquiterpine lactone with a peroxide

group. It is practically insoluble in water and

oil. This drug is less toxic than chloroquine.

It acts on the erythrocytic phases. Its action on

the parasites, which is extremely fast, differs

from that of chloroquine and folate inhibitors


(13). They are thus active against parasites

resistant to chloroquine and antifolate drugs.

2.11 Antibiotic: was shown as tetracyclines, are

often used in conjunction with other drugs to

combat chloroquine resistant falciparum malaria.

Plasmodium protein synthesis appears to be

eukaryotic, and is insensitive to chloramphenicol,

but affected by cycloheximide. It has been

suggested that antibiotics such as tetracycline

act on the mitochondrial ribosomes of the

parasite, inhibiting protein synthesis.

Macrolides such as erythromycin seem to inhibit

autophagic vacuole formation, thus potentiating

the action of chloroquine (14). Resistance to

these compounds is not a current problem.

3. Administration of Drugs

Drugs are given orally except where complications

exist, in which case they are given parenterally.

Parenteral therapy is required when oral administration is

impossible or when the blood infection is heavy and rapid

control of parasites is essential. The indications are the

same for all drugs, and include intractable vomiting;

vascular collapse (shock); coma or delirium; hyperpyrexia;

hyperparasitaemia; other forms of destructively malaria

(15).


4. Plasmodium falciparum Drug Resistance

Resistance has been defined as the ability of parasite

to survive and/or multiply in a concentration of a drug

equal to or higher than that attained by normally

recommended dosage and within the limits of the tolerance of

the subject.

Drug resistant malaria has become one of the most

important problems in malaria control in recent years.

Resistance in vivo has been reported to all antimalarial

drugs except artemisnin and its derivatives (16). Drug

resistance necessitates the use of drugs which are more

expensive and may have dangerous side effects. In some

parts of the world, artemisnin drugs are the first line of

treatment, and are used indiscriminately for self treatment

of suspected uncomplicated malaria- thus is can be expected

to see malaria forms resistant to artemisnin soon according

to WHO. The areas most affected by drug resistance are the

Indo-Chinese peninsula and the Amazon region of South

America.

The problem of drug resistance can be attributed

primarily to increased selection pressures on Plasmodium

falciparum in particular, due to indiscriminate and

incomplete drug use for self treatment (16). In areas such

as Thailand and Vietnam, mosquitoes of the Anopheles dirus and

Anopheles minimus species spread the drug resistant parasites.


These mosquitoes adapt their biting activity to human

behavior patterns, and maintain intense transmission.

Drug resistance is found most commonly in areas of

unstable malaria, or where the drug has been or is being

misused; it has developed in areas in which the antimalarial

drug has been added to table salt for control purposes. It

also occurs in areas in which there is no evidence of misuse

(or even the use) of the relevant drug (14). It can be

produced experimentally in rodent and simian malaria by

repeated drug challenge.

Resistance develops more easily against the

schizonticides proguanil and pyrimethamine, which block the

folic acid: folinic acid cycle of the parasite that 4-amino-

quinolones and mepacrine, which block both the nucleic acid

and the glycolytic cycles. Resistance to quinine is so far

minimal.

The following graph demonstrates the cure rate of drugs

at present and show the decreasing trend in every drug use,

which mean a high level of drug resistant has been occurred.


Figure 2.6: The graph demonstrates the decreasing cure rate

in every drug use in nowadays (16).

The following antimalarial drugs are being widely

resisted by malarial parasites in several strains;

4.1 Resistance to Proguanil and Pyrimethamine

Proguanil are widely used in past two decades, but

the resistances of the parasites were increasingly

existed, thus the drug is not popular in nowadays and was

not used as much in Thailand (14). However, parasites

resistant to Proguanil are usually resistant to

Pyrimethamine (which derived from a metabolite of

Proguanil) and vice versa (15). These drugs act by

sequential inhibition of enzymes of folate metabolism.

Resistance to these drugs has developed over the past 30


years and is now wide spread. Resistance develops very

rapidly and remains stable due to a single point mutation.

The mechanism of resistance to these drugs involves

modification of drug transport systems, increased

synthesis of blocked enzymes, increase in drug

inactivating enzymes and the use of alternative pathways.

Resistance is seen for vivax and falciparum (17). Hence

these drugs may not be of any benefit in complicated

malaria.

4.2 Resistance to the 4-aminoquinolines

Resistance to chloroquine was first noted in

Brazil and Venezuela and shortly afterwards in

Thailand. It is now common in most Southeast Asian

countries, and it is also now appearing in Africa (16).

According to Maegraith (1984), he stated that the

grades of resistance to therapeutic drug dosage are

classified as follows:

Sensitivity: Clearance of asexual erythrocytic

parasites within 7 days of beginning of treatment. No

recrudescence.

Resistance: R I: Clearance of asexual erythrocytic

parasites as in sensitivity, followed by recrudescence

within 28 days


R II: Marked reduction in number of asexual

parasites, without clearance, followed by

recrudescence.

R III: No reduction of asexual parasitaemia.

It should be noted that resistance is graded in

terms of the effect of the drug on the parasites in the

peripheral blood, not on the effect on clinical signs.

In the non-immune these aspects run in parallel. In

the semi-immune clinical relief may be produced without

great reduction in parasitaemia (17).

4.3 Resistance to Primaquine

This drug has primarily been used against

gametocytes and hypnozoites. It has been suggested

that the drug works by inhibiting the electron

transport chain of the parasite, though, as is so often

the case with questions concerning the precise

metabolic interactions, this is uncertain. Neither is

it certain as to whether it is the drug itself or

derived metabolites which have the desired effects

(18). There is no evidence that gametocyte resistance

exists, but if the drug is used against schizonts, then

resistance is rapidly attained (17). The surviving

resistant parasites had increased numbers of

mitochondria suggesting that the resistance mechanism


involves the production of extra organelles to

compensate for the damage caused by the drug.

4.4 Resistance to Sulfonamides

Parasites which become resistant to sulfonamides

must bypass the metabolic step at which para-

aminobenzoic acid (pABA) is incorporated into

dihydropterate. Sulfonamide drugs work by inhibiting

pABA, which is needed to synthesis the dihydropterate

which is an intermediate compound in the synthesis of

tetrahydrofolate. Tetrahydrofolate derivatives serve

as donors of one carbon compounds in a variety of

essential biosynthetic pathways. Little is known about

this side of parasite metabolism, or the exact

mechanisms of resistance – though resistance is clearly

stable, transmissible, and prolific (18). The

resistance seems to be present in all stages of the

parasite metabolism. It is possible that gene

amplification is the mechanism by which the metabolic

block of a pABA inhibitor is overcome (19).

4.5 Resistance to Chloroquine and related compounds

It is known that chloroquine mediates its effects

on the haemoglobin metabolism of malaria parasites,

perhaps preventing the neutralization of the toxic

ferriprotoporphyrin IX(16). Resistant parasites seem


unable to produce haemozoin, but they are still able to

digest haemoglobin. In non-resistant forms, most of

the ferriprotoporphyrin IX is sequestered in haemozoin,

but in the resistant forms, this toxic metabolite seems

to become available to the host cell haemoxygenase

system for elimination (16). In chloroquine- sensitive

malaria, the drug is taken up into food vacuoles, and

it is proposed that here it competes with the

haembinder for the ferriprotoporphyrin IX, to form a

destructive compound (15). A diagrammatic

representation of chloroquine action is shown below.

The Chloroquine is widely used nowadays in every

malarial epidemic area but unfortunately, it is also

widely resisted by numerous strains of Plasmodium

falciparum parasite.

4.6 Resistance to Quinine

Quinine and mefloquine cause blebbing of the

parasite membranes, and causes aggregations of

haemozoin to form. Parasite resistance occurs by

uncertain mechanisms, but is stable and transmissible

(18).

4.7 Resistance to Mefloquine

Sporadic cases of mefloquine resistance have been

reported from Thailand and Kenya (11, 18).


Structurally it is close to quinine and hence cross

resistance with quinine is common. Resistance develops

when the parasite is able to efflux the drug. Even at

the highest dose efficacy of mefloquine is only 50% in

Thailand. Since it is easy to induce resistance for

mefloquine due to its prolonged half life, its use

should be limited, especially since it has cross

resistance to quinine. To prevent development of

resistance to this valuable drug, it has been suggested

that mefloquine should always be used in combination

with another antimalarial, like pyrimethamine or

sulphadoxine.

4.8 Resistance to Artemisnins

In the laboratory, artemisnin resistant forms have

already been demonstrated (18). However it is not confirmed

yet that pfmdr1 locus will affect the drug polymorphism or

not.

In conclusion, it is obvious, then that resistance is

an ongoing problem. By 1973, chloroquine was replaced by

sulfadoxine-pyrimethamine cocktails, but by 1985, this too

was ineffective. Though quinine remains effective, there is

a 50% failure rate unless it is supplemented by

tetracyclines, and compliance with the 7 days regimen is

poor (17). Between 1985 and 1990, the recommended treatment

for malaria in Thailand was mefloquine, combined with


sulfadoxine-pyrimethamine, but by 1990 the cure rate had

fallen to 71% in adults and 50% in children. This treatment

can no longer be used due to resistance (17,18). The future

of chloroquine is not clear, as a recent report (19)

suggests that due to the current absence of chloroquine drug

pressure, chloroquine sensitivity may well be returning.

5. Chemosensitization of Chloroquine Resistance in

Plasmodium falciparum

Research into chloroquine resistance reversal in

Plasmodium falciparum has revealed a widespread range of

functionally and structurally diverse chemosensitizers.

However, nearly all of these chemosensitizers reverse

resistance optimally only at concentrations that are toxic

to humans. Verapamil, desipramine, and trifluoperazine were

shown to potentiate chloroquine accumulation in a

chloroquine-resistance (CQR) strain of P. falciparum, while

progesterone, ivermectin, and cyclosporine A were not shown

to potentiate chloroquine accumulation (20). The

chemosensitizers at concentrates within their therapeutic

ranges in humans displayed an additive effect in

potentiating chloroquine accumulation in the chloroquine-

resistant strain.

The levels of resistance reversal achieved with these

combinations were comparable to those achieved with high


concentrations of the single agents used to enhance the

activity of chloroquine. No chemosensitizer, whether used

singly or in combination, potentiated any change in

chloroquine accumulation or sift in the 50% inhibitory

concentration (IC50) for the chloroquine-sensitive strain.

The use of combinations of chemosensitizers at

concentrations not toxic to humans could effectively reverse

chloroquine resistance without the marked toxicity from the

use of a single agent at high concentrations. This cocktail

of chemosensitizers may serve as a viable treatment to

restore the efficacy of chloroquine in patients with

malaria.

The spread of chloroquine resistance in Plasmodium

falciparum throughout most areas where malaria is endemic has

necessitated alternate treatments for malaria. More

recently, antimalarials such as mefloquine and halofantrine

were developed, but indications are that these are becoming

ineffective as resistance to them spreads (18).

There have been attempts to restore chloroquine

efficacy in vitro and in vivo by using it in combination

with resistance reversers like promethazine and

chlorpheniramine (16, 18). However, these compounds, which

stimulate the uptake of chloroquine by resistant strains and

considerably reduce the 50% inhibitory concentration (IC50),

operate optimally as resistance reversers in vitro only at

concentrations that are highly toxic in vivo. Work with


multidrug-resistant (<MDR) cancer cells has shown that it

is possible to reverse anticancer agent resistance by using

combinations of chemosensitizers at concentrations not toxic

to humans (20). The levels of reversal obtained with these

combinations were comparable to those obtained with the

single agents used at their optimal concentrations.

In P. falciparum, two calcium channel blockers, verapamil

and fantofarone have been shown to act synergistically in

the reversing chloroquine resistance (21). It have been

selected several structurally and functionally diverse

compounds to test chloroquine resistance reversal in

Plasmodium falciparum. Verapamil is known resistance reversers

in Plasmodium falciparum (18, 22). A combination of the

chemosensitizers used at low concentrations was shown to

work as effectively in vitro in reversing chloroquine

resistance as the single compounds used at their optimal

concentrations with chloroquine. This may yet prove to be

an effective way of overcoming the chloroquine resistance

without the toxicity associated with these chemosensitizers

in vivo. Resistance to chloroquine by the malaria parasite

Plasmodium falciparum has been observed in every region where

Plasmodium falciparum occurs (20). The exact mode of action of

chloroquine has not been fully elucidated, but it is

generally accepted that a crucial step in this process is

the binding of the drug to ferriprotoporphyrin IX (heme), a


by-product of hemoglobin degradation which occurs in the

parasite digestive food vacuole (23).

6. pfmdr1 and pfcrt

Chloroquine resistance in Plasmodium falciparum is

associated with mutations in the digestive vacuole

transmembrane protein PfCRT. However, the contribution of

individual PfCRT mutations has not been clarified and other

genes have been postulated to play a substantial role (22).

Using allelic exchange, was shown that removal of the single

PfCRT amino-acid changes the parasites from resistant

strains leads to wild-type levels of chloroquine

susceptibility, increased binding of chloroquine to its

target ferriprotoporphyrin IX in the digestive vacuole and

loss of verapamil reversibility of chloroquine and quinine

resistance(23). It has been indicated that PfCRT mutations

preceding residue 76 modulate the degree of verapamil

reversibility in chloroquine-resistant lines. The K76T

mutation accounts for earlier observations that chloroquine

resistance can be overcome by subtly altering the

chloroquine side-chain length, Simultaneously, these

findings establish PfCRT – K76T as a critical component of

chloroquine resistance and suggest that chloroquine access

to ferriprotoporphyrin IX is determined by drug-protein

interactions involving this mutant residue (24).


A number of studies have contributed to pinpointing the

PfCRT gene as the major determinant of chloroquine

resistance (24, 25). In addition, mutations of the pfmdr1

gene (expressing Pgh1) have been shown to modulate the level

of chloroquine resistance (19), as well as being partially

responsible for the acquired resistance to other drugs such

as mefloquine (23). Currently there are two hypotheses as

to the function of PfCRT in chloroquine resistance. The

first of these proposes that PfCRT actively removes

chloroquine from the digestive vacuole, either as an ATP-

dependent pump or as a secondary active transporter (25).

Alternatively, the “charged drug leak model” proposes that

diprotonated chloroquine (CQ++) leaves the digestive vacuole

via mutated PfCRT passively down its concentration gradient

(26). Theories are in agreement, however, that chloroquine

is transported out of the digestive vacuole and that this is

the key mechanism of chloroquine resistance.


Figure 2.7: Mechanism of reduce Chloroquine mechanism in the

parasites cell. The pfmdr1 and Pgh 1 will active in the area

of heme.

The exact role of the pfmdr1 gene in the emergence of

drug resistance in the malaria parasite Plasmodium falciparum

remains controversial. Pfmdr1 is a member of the ATP

binding cassette (ABC) superfamily of transporters that

includes the mammalian P-glycoprotein family (25). It has

been introduced wild-type and mutant variants of the pfmdr1

gene in the P. falciparum and have analyzed the effect of

pfmdr1 expression on cellular resistance to chloroquine-

containing antimalarial drugs. Parasites transformants

expressing either wild-type or a mutant variant of


erythrocytes P-glycoprotein were also analyzed. Dose-

response studies showed that expression of wild-type pfmdr1

causes cellular resistance to chloroquine in parasites(26).

Figure 2.8: The Pgh1 protein of Plasmodium falciparum.

Polymorphic amino acids are indicated.

Pfmdr1, which codes for the plasmodial homologue of

mammalian mdr genes in P. falciparum, was cloned and

sequenced (25). It is a typical member of the ABC

transporter superfamily, a polypeptide of ~162 kD, with a

conserved structure of two domains consisting of six

predicted transmembrane segments coupled to a nucleotide-


binding fold joined together by a linker region, and has

been termed Pgh1 (P-glycoprotein homolog 1) (26). Pgh1

was subsequently localized to the parasite vacuole

throughout the asexual cycle of the parasite, where is

was postulated to regulate intracellular drug

concentrations (26). It binds ATP and is phosphorylated

extensively on serine and threonine residues by a

calcium-dependent protein kinase (27).

However, almost ten years after the first evidence of

an association between pfmdr1 mutations and chloroquine

resistance was made; newly available transfection methods

in P. falciparum were utilized to definitively demonstrate

that pfmdr1 mutations can modulate resistance levels to

chloroquine (27). Significantly, in the studies of Brey

et. al. (2005), mutations introduced into the

chloroquine-sensitive line D10 were unable to confer

chloroquine-resistance. Nevertheless, introduction of

wild-type polymorphisms into 7G8, a chloroquine-

resistance line, resulted in the reduction of (reversal)

of chloroquine-resistance, suggestion that pfmdr1 although

important in conferring higher levels of chloroquine-

resistance, is not sufficient by itself to confer

resistance. Mutation of wild type pfmdr1 was sufficient

to confer quinine resistance in the sensitive D10

parasite line (27).


7. pfmdr1 and in vitro sensitivity of Verapamil.

Figure 2.9: The chemical structure of verapamil

In general, verapamil (VP) is a weak base and has one

characteristic which is to acting as a reverser of

chloroquine resistance (CQR) in the P. falciparum.

Nonetheless, it also has an extra characteristic as an

intrinsic antiplasmodial activity (28). This activity is

independent to its chloroquine resistance (CQR) reversal

effect as the susceptibility of chloroquine-sensitive

parasites to chloroquine is altered even in the presence

of highly toxic concentration of verapamil (VP), whereas

verapamil alters the susceptibility of chloroquine

resistance (CQR) parasites to chloroquine at both toxic

and nontoxic concentration (28).

The chloroquine resistance (CQR) reversal effect of

verapamil has been attributed to an interaction of the

compound with the P. falciparum chloroquine resistance


transporter (PfCRT) (26, 27), a member of drug-metabolite

transporter superfamily (26) which is localized to the

parasite internal digestive vacuole and is the key

determinant of chloroquine resistance.

Furthermore, in many studies have confirmed that

mutations in the pfmdr1 gene product, P-glycoprotein

homologue -1 (Pgh1), modulate sensitivity to a sort of

antimalarial compounds (25). It has been reported that

polymorphism in pfmdr1 influence the parasite’s

susceptibility to the intrinsic antiplasmodial effect of

verapamil (28).

The chloroquine-sensitive strains, D10 and the

chloroquine-resistant, 7G8 strains with pfmdr1 loci

altered as described previously were used in the model

experiment (Hayward et. al. 2005). Subsequently, the

introduction of either one or three of the four 7G8

mutations into the pfmdr1 gene of D10 parasite

significantly increased verapamil sensitivity with

respect to the D10- mdr10 transfectant, a control

transfectant with a wild type pfmdr1 gene. Further

analysis shows that the pattern of relative sensitivity

of these transfectants to verapamil correlates

significantly with the pattern of sensitivity to

mefloquine (MQ) and halofantrine (HF) in their previous

study (26). The result suggests that the specific

mutation in pfmdr1 mediate sensitivity to these compounds


via a common mechanism. There is no difference in the

level of Pgh1 expression- a parameter which has been

shown to affect mefloquine sensitivity- among these

strains (27).

However, the similarity of verapamil and mefloquine

sensitivity profiles of these P. falciparum transfectants

invites comparison with the interaction of these drugs

with the P-glycoprotein. It also shown in the previous

study that mefloquine has better ability to bind the P-

glycoprotein with better affinity and also shown to be

more potent inhibitor of P-glycoprotein than verapamil.

From the experiment, it was shown that while these

transfectants respond to verapamil and mefloquine with

the same pattern of sensitivity verapamil is far less

potent than mefloquine (26). As a result, these data

prompt the hypothesis that verapamil and mefloquine

interact directly with Pgh1 and the mutation of the

interest here affects the sensitivity of parasites to

verapamil and mefloquine by influencing this interaction

(27).

In conclusion, the data in the model research

demonstrate a role for Pgh1 in mediating parasite

sensitivity to the intrinsic antiplasmodial effects of

verapamil. The fact that mutations in pfmdr1 have the

same effects on parasites sensitivity to the lipophilic

drugs verapamil, mefloquine and halofantrine suggests a


common mechanism of resistance to these drugs, possibly

through a direct interaction with Pgh1 (28).


CHAPTER III

MATERIALS AND METHODS

1. Materials

1.1. Parasite isolates: both wild type and mutation

1.1.1. Wild type isolates: PCM 4, BC 5, BC 6, PCM 14,

BC 13, BC 21, BC 24, BC 28, BC 35 and J13.,

1.1.2. Mutation isolates: BC 1, K14C, M 12, TM 5, TM 6,

T 994, PCM 8, BC 11, KS 17 and RN 3

1.2. Uninfected red blood cells

1.3. Cryotubes

1.4. Liquid nitrogen refrigerator

1.5. Complete Medium

1.6. RPMI + Gentamycin

1.7. RPMI for wash

1.8. 3.5% NaCl solution

1.9. HEPES

1.10 . Serum

1.11. 5% Sorbital

1.12. Incubator for cell culture

1.13. Centrifuge


1.14. Autoclave

1.15. Glass slides

1.16. Microscope

1.17. Cell culture flasks 50 ml

1.18. Pasteur pipette

1.19. Autopipette

1.20. Pipette (0.1, 1, 5, 10, 20, 100, 200, and 1,000

ml)

1.21. Pipetteman

1.22. Pipette tips (1, 100, 1000 µl)

1.23. Laminar airflow cabinet

1.24. Mixed Gas 5%CO2, 5%O2, 90%N2

1.25. Verapamil

1.26. [3H]hypoxanthine

1.27. Microtitre plates

1.28. Printed Filtermat A

1.29. Dynatech Automash 2000 semi-automatic cell

harvester

1.30. 6 ml polypropylene scintillation fluid vials

(LIP, U.K.)

1.31. Optiphase ‘Safe’ scintillation fluid (LKB, U.K.)

1.32. Scintillation Counting Machine

2. Methods

2.1 Culture system for parasite maintenance


All parasite isolates of Plasmodium falciparum used in this

thesis were cultured by an adaptation of the methods of

Trager and Jensen (1976) and Jensen and Trager (1977). All

culture work was carried out using standard aseptic technique

in an Envair class II laminar flow safety cabinet. All

consumable containers such as culture flasks, centrifuge

tubes and universal bottles were of pre-sterilised disposable

plastic. All glassware was autoclaved at 120 oC, 15

atmospheres for 15 minutes prior to usage. All solutions were

sterilised either by filtration through a 0.2 m acrylic

filter (Gelman Sciences Inc., U.K.) or by autoclaving. Hands

were rinsed regularly with 70 % ethanol when working in the

laminar flow safety cabinet in order to minimise

contamination. Basic culture techniques are outlined below.

2.1.1 Parasite isolates

Twenty isolates of P. falciparum were used in the studies

described. These isolates included the Wild type isolates:

PCM 4, BC 5, BC 6, PCM 14, BC 13, BC 21, BC 24, BC 28, BC

35 and J13.,


Mutation isolates: BC 1, K14C, M 12, TM 5, TM 6, T 994,

PCM 8, BC 11, KS 17 and RN 3 are kindly provided by Dr.

Mathirut Moongthin, Department of Parasitology,

Pramongkutklao College of Medicine.

The original source and CQ sensitivity status of these

isolates is summarised below (Table 2.1.1.1). Isolates with a

CQ IC50 of less than 80 nM are defined as susceptible and

isolates with a CQ IC50 more than 80 nM are defined as

resistant.

Isolate Source Pfmdr1

K14C Thailand Mutation

BC1

Unknown Mutation

M 12 Thailand Mutation

TM 5 Thailand Mutation

TM 6 Thailand Mutation

RN 3 Thailand Mutation

T 994 Thailand Mutation

PCM 8 Thailand Mutation

BC 11 Thailand Mutation

KS 17 Thailand Mutation

PCM 4 Thailand Wild Type

BC 5 Thailand Wild Type



PCM 14 Thailand Wild Type






Isolate Source Pfmdr1

J 13 Thailand Wild Type

Table 2.1.1.1 Source and CQ sensitivity of the isolates of P.

falciparum used in these studies.

2.1.2Culture Medium

Culture medium for malaria parasites was prepared as

follows: 10.43 g of lyophilised RPMI 1640 containing L-

glutamine (Gibco, U.K.) and 2.0 g of sodium hydrogen

carbonate (sodium bicarbonate; Sigma, U.K.) was dissolved in

1 L of distilled water. After the solution was stirred

continuously for 3-5 h using a magnetic stirrer, the stock

medium was sterilised by filtration through a 0.2 m acrylic

filter (Gelman Sciences Inc., U.K.) using a Millipore (U.K.)


peristaltic pump. The stock medium was stored, at 4 oC in 500

ml aliquots for up to 2 weeks.

To check for contamination the stock medium was

incubated at 37 oC for 24 h before use. Contamination was

characterised by an increase in turbidity of the medium

and/or by a colour change of the medium from red/orange to

yellow (brought about by an increase in the acidity of the

medium as a consequence of the lactic acid produced by the

contaminating micro-organisms). Contamination was confirmed

by visual analysis of a thin 10 % Giemsa stained blood film

of the suspect parasite culture, by light microscopy (see

Section 2.1.8)

Complete culture medium was prepared by adding 12.5 ml

of a 1 M pre-sterilised HEPES (N-2-hydroxyethylpiperazine-N'-

2-ethanesulfonic acid) buffer solution (Sigma, U.K.), 0.5-1

ml of a 10 mg ml-1 gentamicin solution (Gibco, U.K.) and 50 ml

of pooled human AB serum (see Section 2.1.4), to each 500 ml

aliquot of stock medium. This complete medium was then

incubated at 37 oC for 24 h prior to use in order to check

for contamination. Contamination was characterised as


described above. Any unused complete medium was discarded

after 1 week to avoid the effects of age related medium

deterioration.

2.1.3Uninfected erythrocytes

Figure 3.1: The erythrocyte for cultivated the parasites.

Human group O Rhesus positive fresh whole blood

(obtained no longer than 48 h after collection from donors)

was kindly supplied by the Pramongkutklao College of

Medicine, Bangkok. This blood (consisting of bags of

irregular volume, unsuitable for transfusion) was supplied in

citrate-phosphate-dextrose bags and had been tested negative


for anti-HIV and anti-hepatitis B antibodies. On arrival, the

fresh blood was transferred aseptically to sterile 250 ml

culture flasks and stored at 4 oC for up to three weeks.

Prior to usage, the serum and buffy coat were removed

using a pre-sterilised cotton plugged Pasteur pipette after

10 ml aliquots of whole blood were centrifuged aseptically

(2000 x g, 10 min). The remaining packed erythrocytes were

washed three times by resuspending in 10 ml of sterile

phosphate buffered saline, pH 7.2 (8.5 g NaCl; 1.07 g Na2HPO4;

0.39 g NaH2PO4; in 1 L distilled water). After each wash, the

erythrocytes were sedimented by centrifugation (2000 x g, 10

min) and the supernatant discarded. After washing was

complete, erythrocytes were stored as packed cells at 4 oC

for up to 1 week.

2.1.4Serum

Human AB serum was kindly supplied by the Pramongkutklao

College of Medicine, Bangkok. 100 - 250 ml bags of serum,

produced from a single unit of whole blood were supplied

weekly. In order to minimise batch variation effects, 8 - 10


bags of serum were pooled at a time and stored in 50 ml

aliquots at -20 oC until used.

2.1.5Gas phase

It has been shown that prolonged parasite growth

requires an atmosphere with a lower O2 concentration and a

higher CO2 concentration than atmospheric air (25). The gas

phase used throughout in these studies was composed of 93 %

N2, 3 % O2 and 4 % CO2 (prepared and supplied by British

Oxygen Special Gases, Thailand).

Culture flasks were gassed aseptically, inside the

laminar flow safety cabinet as follows: The gas from the

cylinder was delivered to the laminar flow cabinet via a

length of pre-sterilised silicone rubber tubing. The gas then

passed through a 0.2 m pore size acrylic filter (Gelman

Sciences Inc., U.K.), into a further length of sterile

silicone rubber gas line terminated with another 0.2 m

acrylic filter. The terminal filter was replaced at the

beginning of each day and the gas line was sterilised every

two weeks. Culture flasks were gassed via individual,


sterile, 19 G needles (Beckton-Dickinson, U.K.) fitted to the

terminal 0.2 m acrylic filter. A fresh sterile 19 G needle

was used to gas each individual flask.

2.1.6Parasite cultivation procedure

Cultures were maintained in pre-sterilised plastic

flasks (Nunclon, U.K.) of 50 or 200 ml capacity depending on

the amount of parasite material required. The haematocrit or

cell density in these flasks varied between 1 % and 10 % but

was most commonly 2 %.

Cultures were initiated by seeding a red cell/complete

medium suspension with parasitised red cells from either

another culture flask or parasitised cells revived from

cryopreserved stocks (see Section 2.1.7) to give the required

haematocrit. Cultures were usually initiated at about 0.1 %

parasitaemia and 2 % haematocrit, however if parasites were

required quickly, higher starting parasitaemias were employed

(up to 2 %).

When parasitaemias were low (less than 1.5 %) culture

medium was changed every 48 h, however at higher


parasitaemias the medium was changed every 24 h. The

procedure for this was as follows: spent medium was removed

aseptically from above the static cell layer with a sterile

cotton plugged Pasteur pipette and discarded. Pre-warmed

fresh complete culture medium was then added in volumes of 15

ml to flasks of 50 ml capacity and 50 ml to flasks of 200 ml

capacity. These flasks were then gassed as described above

(see Section 2.1.5). The duration of gassing was 30 s for

flasks of 50 ml capacity and 60 s for flasks of 200 ml. The

culture flasks were then placed in an incubator at 37 oC.

Flasks were subcultured when the target parasitaemia had

been reached (up to 20 %). The subculturing procedure was as

follows: fresh red cell/medium suspension at the required

haematocrit was added to a new flask labelled with the name

of the isolate, the date of the subculture and the subculture

number. Most of the medium was removed from the donor flask

and the cell layer was resuspended in the remaining medium. A

small aliquot (approx. 10 µl) of this suspension was used to

seed the new culture flask at the required starting

parasitaemia. The new culture flasks were then gassed and


incubated as described above. The remainder of the original

culture was either processed for experimental use,

cryopreserved as described below (see Section 2.1.7), or

discarded.

2.1.7Cryopreservation and retrieval of parasite cultures

Two cryopreservation techniques were used in these

studies, during 1994-1996 the cryopreservation was based on

the method of Wilson et al. (1977). This procedure is as

follows: cultures of high parasitaemia (> 5 %), predominantly

at ring stage, were transferred aseptically to a sterile

centrifuge tube and centrifuged (2000 x g, 10 min). The

supernatant was removed and fresh medium was added to give a

50 % haematocrit. An equal volume of ice cold 20 % DMSO (in

PBS) was then added to this cell suspension and the mixture

was aliquoted quickly into sterile 1.8 ml cryotubes (Nunclon,

U.K.), in aliquots of 0.5-1 ml per tube. These cryotubes were

labelled with the isolate name and date, before being plunged

into liquid nitrogen. When frozen the tubes were transferred

to a liquid nitrogen refrigerator for storage. After 1997 the


modified method of Rowe et al. (1968) was used due to rapid

recovery of parasites after retrieval. The cryoprotectant was

prepared by adding 70 ml of glycerol to 180 ml of 4.2%

sorbitol in physiological saline. An equal volume of

cryoprotectant was added to the parasitised packed cells and

allowed to equilibrate for 5-10 minutes at room temperature;

the cryotubes were then plunged into liquid nitrogen and

transferred to liquid nitrogen storage.

Cryopreserved cultures from both methods were retrieved

as follows: cryotubes were removed from the liquid nitrogen

refrigerator and thawed at 37 oC. The contents of the tube

were then aseptically transferred to sterile centrifuge tubes

and centrifuged (2000 x g, 10 min). The supernatant was

removed and the pellets resuspended in an equal volume of ice

cold 3.5 % NaCl. The tubes were then re-centrifuged (2000 x

g, 10 min), the supernatant was discarded and the pellets

were washed by resuspending in complete culture medium and

followed by centrifugation as before. The supernatant was

removed and the pellets resuspended in 15 ml of complete

culture medium made up to the required haematocrit with


washed uninfected erythrocytes. The contents of the tubes

were then placed in sterile 50 ml culture flasks, gassed and

placed in an incubator at 37 oC.

2.1.8Routine monitoring of parasitaemia

At the beginning of each day thin blood films were

prepared from each culture flask by spreading a drop of

cultured cells on a new, clean, glass microscope slide. Films

were then fixed for 5 s in methanol and placed into a 10 %

solution of Giemsa stain (BDH, U.K.) in distilled water,

buffered at pH 7.2, for 20 min. Blood films were then washed

in tap water, dried and examined under oil immersion at x

1000 magnification on a light microscope (Zeiss, Germany).

The parasitaemia was calculated as the number of

infected cells expressed as a percentage of the total number

of cells counted in 5 - 10 fields of the blood film.


Figure 3.2: Blood smear use for calculate and observe the

trophozoite phase of parasites.

2.1.9Synchronisation of parasite cultures

Highly synchronous cultures were used throughout the

studies described in this thesis. Parasites were synchronised

regularly by the method of Lambros and Vandenburg (1979).

Cultures with a high proportion of ring stage parasites (this

technique selectively lyses the later stage parasites which

are more permeable to sorbitol, causing them to swell and

eventually lyse) were transferred aseptically to sterile

centrifuge tubes and centrifuged (2000 x g, 10 min). The

supernatant was then discarded and the pellets resuspended in


5 volumes of 5 % aqueous sorbitol. The solution was left to

stand at room temperature for 20 min and then re-centrifuged

(2000 x g, 10 min) and the supernatant removed. The pellets

were washed by resuspending in complete medium followed by

centrifugation (2000 x g, 10 min). Medium was discarded and

the pellets were resuspended in complete medium and the

suspension placed back into culture for 48 h before use.

2.1.10 Stage specific parasite isolation

Late trophozoite and schizont stage parasites were

concentrated and separated from early trophozoite, ring stage

parasites and uninfected red cells by a method developed by

Kramer et al, (1982). The technique relies on density gradient

centrifugation through Percoll and is outlined below:

Concentrated Percoll (Sigma, U.S.A.) was diluted 9:1 with

sterile 10X concentrated PBS. This isotonic 90% Percoll

solution was further diluted to a 63% solution by adding 1X

concentrated PBS (pH 7.2), 32 ml of 63% Percoll solution was

then dispensed aseptically into a sterile 50 ml round bottom

centrifuge tube. After 1 ml of packed cell from culture was


mixed with the Percoll in the centrifuge tube, the tube was

centrifuged at 39000 X g for 30 min. Four distinct zones

could be seen after centrifugation; zone 1 (top) contained

only pigment and cellular debris, zone 2 and 3 contained a

highly concentrated mixture of late trophozoites and

schizonts and zone 4 contained ring and early trophozoite

stage parasites and uninfected erythrocytes. The parasites

from a desired zone were removed from the gradient with a

sterile Pasteur pipette, washed twice by centrifugation at

600 X g for 5 min and a blood film was made and stained with

Giemsa.

2.1.11 Decontamination of parasite cultures

From time to time cultures would become infected with

bacterial or fungal growth. Decontamination of cultures

infected with bacteria was attempted using one of two

methods:

a) Penicillin-streptomycin-neomycin (Gershon, 1985).

Cultures infected with low levels of bacteria (less than one

bacterium per field on a Giemsa stained thin blood film) were


decontaminated by first washing the cells by centrifugation

(2000 x g, 10 min) in complete medium and then replacing the

medium with that containing penicillin-streptomycin-neomycin

solution (Gibco, U.K.) at a dilution of 50 : 1 (v/v).

Cultures were treated until no bacteria were visible on daily

blood films, and then for a further three days.

b) Chloramphenicol (Yayon et al., 1984c). Cultures were

first washed by centrifugation as above. Fresh medium

containing chloramphenicol (Sigma, U.K.) at 0.1 mg ml-1 was

added and the cultures were incubated at 37 oC for 4 h.

Cultures were then washed by centrifugation, resuspended in

complete medium without antibiotic for 24 h at 37 oC and then

treated with chloramphenicol medium as before. Cells were

then washed and put back into culture.

Low level fungal contamination was treated using the

method of Yayon et al. (1984c). Cells were washed in complete

medium, centrifuged (2000 x g, 10 min) and the medium was

then discarded. The cell pellet was resuspended in medium

containing 12-25 g ml-1 nystatin (Mycostatin, Squibb, U.K.)

for 4 h. After this period, the cells were again washed in


complete medium, as described above, followed by resuspension

in medium containing 6 g ml-1 nystatin. This concentration

of drug was maintained in the culture medium until

contamination was no longer visible and then for a further

three days.

2.2 In vitro parasite drug sensitivity assay

2.2.1Technique

Throughout these studies the in vitro activity of a number

of different compounds was assessed against various isolates

of P. falciparum. The method employed was an adaptation of the

standard 48 h microdilution assay developed by Desjardins et

al. (1979). This method relies on the ability of P. falciparum to

incorporate the nucleic acid precursor, hypoxanthine.

Incorporation of radiolabelled hypoxanthine

([3H]hypoxanthine) is therefore used as a marker of parasite

growth. Details of the procedures involved are outlined

below:

2.2.2Preparation of drug solutions


In most cases, the drugs used in this study were

dissolved in solvents (H2O, EtOH, MeOH, DMSO or a

combination), at a concentration of 10-2 M. These stock

solutions were then serially diluted with complete medium

(without hypoxanthine) to give the required range of drug

concentrations for each assay. The final concentration of

organic solvent in the assay plates was always less than 0.1

% which was shown to have no effect on parasite growth.

Figure 3.3: Serial drug dilution in different concentration.

2.2.3Preparation of parasites

Parasites were synchronised at ring stage (see Section

2.1.9), 48 h prior to use. Parasitaemia of these

predominantly ring stage parasites was assessed as described

above (see Section 2.1.8). The cell suspension was then


centrifuged (2000 x g, 10 min) and the supernatant discarded.

The packed cells were diluted with washed fresh erythrocytes

to give a final parasitaemia of 1%. These cells were then

washed twice in sterile PBS, followed each time by

centrifugation (2000 x g for 10 min). Finally the packed

cells were resuspended in complete medium (without

hypoxanthine) to give a final suspension of 1% parasitaemia

at 20% haematocrit.

2.2.4Preparation of microtitre plates

Figure 3.4: Microtitre plates for testing the sensitivity of

each strain on verapamil.


The microtitre plates used in this study were of the 96-

well individually wrapped, pre-sterilised plastic type

(Microwell, Nunclon, U.K.). Wells are arranged in 8 columns

(labelled A through to H), each containing 12 rows (numbered

sequentially from 1 through to 12). Extreme care was taken

when preparing the plates to avoid contamination. The plates

were prepared as follows: each assay was performed in

triplicate, using adjacent wells, on one half of a microtitre

plate. The outer wells of the plate (columns A and H, and row

1) were not used for assay purposes. The reason for not using

these outer wells was that previous workers have shown that

these wells do not support good parasite growth (Gershon,

1985).

Each assay was performed in triplicate on three adjacent

rows in the plate (for example columns B, C and D) leaving

room for 2 assays in plates. Drug-free complete medium

without hypoxanthine was added to wells in rows 6,7 (the

parasitised control wells) and 12 (the unparasitised control

or radioactive background control wells) in 100 l volumes

using a 100 l automatic pipette (Gilson Pipetteman, Gilson,


U.K.) with the appropriate pre-sterilised tip. Drug dilutions

in complete medium without hypoxanthine were added to wells

in rows 2 - 5 and 8 - 11 (2 being the highest and 11 being

the lowest concentration). Culture inoculum prepared as

described in Section 2.2.3 was added to each occupied well in

rows 2-11 in 10 l volumes, using a 10 l Gilson pipette with

presterilised tip. The total volume of cell/medium suspension

in each well was 110 l at a 1-2% haematocrit. Uninfected red

cell suspension (20% haematocrit in complete medium without

hypoxanthine) was added to wells in row 12 in 10 l volumes

as the unparasitised control or radioactive background

control wells).

Once completed, the plates were covered with their own

sterile lids and placed in a modular incubation chamber

(Flow, U.K.), gassed for 5 min in the laminar flow cabinet

and incubated at 37 oC for 24 h. At the end of the 24 h

incubation period the plates were removed from the chamber

and radiolabelled hypoxanthine was added to each well as

described below:


2.2.5Preparation and addition of [3H]hypoxanthine

The radiolabelled hypoxanthine used throughout these

studies was supplied by NEN (U.S.A.) in 5 mCi aliquots made

up in 5 ml of sterile water to give a 1 mCi ml-1 solution.

The specific activity of each batch of [3H]hypoxanthine was

approximately 50 Ci mmol-1.

An aliquot of this 1 mCi ml-1 solution was diluted ten

fold with complete hypoxanthine free medium to give a 100 Ci

ml-1 solution. At the end of the initial 24 h incubation

period, 5 l of this radioactive-labelled solution was added

to each well of the assay plate using an automatic pipette

(Gilson Pipetteman, Gilson, U.K.) and the appropriate sterile

tip. Each well received 0.5 Ci hypoxanthine. Plates were

then shaken gently to ensure that the contents of each well

were thoroughly mixed. The plates were placed back in the


modular incubation chamber, gassed for 5 min and incubated at

37 oC for a further 24 h.

2.2.6Harvesting of assays

After the second 24 h incubation period was completed,

the plates were removed from the incubation chamber. Plates

were shaken gently to ensure thorough mixing of the contents

of each well and harvested using a Dynatech Automash 2000

semi-automatic cell harvester (1994-1995). This cell

harvester works by flushing the entire contents out of each

well of the assay plate with distilled water under reduced

pressure and depositing them on a glass fibre filter mat in

circles of 1 cm diameter. After 1996 Printed Filtermat A

(Wallac, Finland), a glass fibre filter for 1450 MicroBetaTM

was used and the assay plate was harvested by a Tomtec March

III M semi-automatic harvester. These filter mats were

partially dried under reduced pressure, by a stream of air

and then removed from the harvester, allowed to dry fully in

an oven at 60 oC prior to scintillation counting.

2.2.7Scintillation counting


Figure 3.5: The Scintillation counting machine.

Liquid scintillation counting was employed to measure

the amount of radioactivity incorporated by individual groups

of parasites as follows: once dry, the filter discs on the

filter mat, corresponding to each well of the assay plate,

were removed and placed in 6 ml polypropylene scintillation

insert vials (LIP, U.K.). 4 ml of Optiphase 'Safe'

scintillation fluid (LKB, U.K.) was then added to each of the

vials, which were sealed with flush fitting plastic caps. The

vials were placed into scintillation racks ready for

scintillation counting. The machine employed for assessment

of the radioactive content of each of the vials was an LKB

Rackbeta 1219 scintillation counter.

From 1996 the radioactivity was measured by 1450

MicroBeta Trilux liquid scintillation and luminescence


counter (Wallac, Finland), samples were prepared as follows:

MeltiLexTMA (Wallac, Finland) a melt-on scintillator sheet was

placed on top of the dry filter mat in a sample plastic bag

(Wallac, Finland), this was then heated using a 1495-021

Microsealer (Wallac, Finland), each sample was then placed

in a cassette ready for counting.

2.2.8 Analysis of data

Parasite growth in the presence of increasing

concentrations of drug was assessed by comparing the level of

radiolabelled hypoxanthine incorporation in the presence of

drug with that of controls containing no drug. The amount of

radioactivity incorporated was measured as disintegrations

per minute (dpm). For each assay mean dpm values were

calculated for parasitised controls, unparasitised controls

and for each triplicate group of wells containing drug.

Following subtraction of unparasitised control values,

percentage parasite growth at each drug concentration was


calculated from comparison with parasitised controls (which

represented 100 % growth).

Data was represented graphically in the form of a log

dose response curve. This graph is produced by plotting log

drug concentration on the abscissa against the percentage

parasite growth on the ordinate axis. Representative log dose

response curves were plotted for each drug using the Grafit

computer programme package, Erithacus Software Ltd., Staines,

U.K. This programme automatically calculates drug IC50 via

interpolation of the log dose response graph at the 50 %

growth mark on the ordinate axis. These IC50 values were used

as a measure of drug potency to compare the activity of the

compounds tested throughout these studies.

CHAPTER IV


RESULT

1. Inhibitory Concentration (IC 50) Table

Wild type MutationStrains IC 50 (µM) Strains IC 50 (µM)PCM 4 25.7 BC 1 29.78BC 5 16.04 K 14C 27.62BC 6 28.71 M 12 35.54

PCM 14 28.21 TM 5 29.99BC 13 9.74 TM 6 36.23BC 21 10.88 T 994 36.78BC 24 24.58 PCM 8 22.72BC 28 28.89 BC 11 9.92BC 35 40.46 KS 17 22.14J 13 20.38 RN 3 20.87

Table 4.1: The average IC 50 of both P. falciparum wild type

isolates and mutation isolates containing pfmdr1 in the

concentration of µM.

The tables below demonstrate the sample of parasite

growth in each verapamil concentration growth of IC 50 that

calculated from isolates.

VP IC 50= 30.9981 m= 3.3249Conc. J 13

02889 2739 2922

2797.5

2622 2850 2763

1 1783 1919 2003 19020.6797736

07

5 2060 2042 2006 20360.7277926

72


10 2117 2015 2009 20470.7317247

54

25 1318 1243 1462 13410.4793565

68

50 306 339 306 3170.1133154

6

75 138 159 135 1440.0514745

31

100 132 72 99 1010.0361036

64

250 57 48 54 530.0189454

87

Table 4.2: The relationship between verapamil concentration

and parasite growth using J13 isolates.

VP IC 50= 28.2198 m= 2.4957Conc. PCM 14

04686 5154 5010

5372.33

5855 5671 5858

1 2468 2144 1940 21840.40652752

2

5 2586 2607 2111 24350.45318635

8

10 2420 2258 2297 23250.43277311

7

25 1252 1381 1181 12710.23664468

450 384 540 597 507 0.09437246

75 150 258 297 2350.04374265

9100 75 75 144 98 0.01824162

250 69 96 81 820.01526339

6

Table 4.3: The relationship between verapamil concentration

and parasite growth using PCM 14 isolates.


2. Graph of Inhibitory Concentration (IC50) in the unit

of µM.

The representative graphs demonstrate the growth curve of

parasite isolates that susceptible to verapamil in each

increasing concentration.

Figure 4.1: The effect of increasing verapamil concentrationon the growth of J 13 parasite isolates.

VP J 13

VP PCM 14


Figure 4.2: The effect of increasing verapamil concentrationon the growth of PCM 14 parasite isolates.

Note: These tables were calculated by Grafit

program. They were plotted by

adding the IC50 value of each strain.

IC 50 pfmdr1 Wild Type Mutation

Minimum 9.7437 9.9168 Maximum 40.4657 36.7895

Mean Value 24.6614 29.5909p- value=0.353

Std.Deviation 10.2322 9.9244 SD. Error

Mean 2.9538 3.5088

Table 4.4: Statistical Analysis of both wild type and

mutation with the

p- Value = 0.300

Values are the mean +/- SD concentration of drug

that inhibits parasite growth by 50% (IC50) (nM)

derived from at least three separate assays

performed at a hematocrit of 1% and a parasitemia of

1%


There is no significant different of VP IC50 between

wild type isolates and mutation isolates

(Independent t-test, p>0.05)

MutationWild typePfmdr1

40.00

30.00

20.00

10.00Verap

amil IC 50

(micr

omola

r)

Graph boxplot of Analytical data

Graph 4.1: Boxplot graph shows the different means and analytical statistic data.


CHAPTER V

DISCUSSION

According to the statistics data, the p-value on the

experiment is equal to 0.353, it is indicated that there is

no significant different between wild type and mutation

isolates on innate antiplasmodial activity and sensitivity

of verapamil in Plasmodium falciparum isolates. Thus, it

completely against ,the previous studies of Hayward et al,

(2005), which indicate that there is significant different

between 2 strains. Hence, this study definitely disparate

the previous studies of Hayward et al. (2005) that the

pfmdr1 does not altered the sensitivity in both mutation and

wild type isolate. The specific mutation in pfmdr1 does not

mediate sensitivity to verapamil via common mechanism.

In general, scientists recognize that a fix 5 µM

concentration is the best concentration which can


effectively reverse the chloroquine resistance activity in

P. falciparum which less than 10% of parasites will be

exterminated. It is used as fix concentration for every

single isolate in nowadays. Nonetheless, the inhibitory

concentration of each isolate does not evidently know yet.

Thus, this study revise the IC50 of these twenty isolates in

order to enhance the further experiment since the standard

IC50 of each isolate are unknown yet. Knowing the exact dose

of IC 50 in each strain will elevate the process of

experiment to be faster and have a certain result. It is

advantage to identify the correct IC50 of each strain

because in the in vivo and in vitro sensitivity and

chemosentisisation experiment, the concentration of 5 µM or

higher, might toxic some strain and might exterminate the

parasite up to 50% and with this rate the sensitivity

testing will not effective at all.

However, since the parasite isolates of this experiment

were received from the nature, it is suspected that there

might be other genes rather than pfmdr1 alone does associate

and alter the sensitivity of verapamil in Plasmodium falciparum

isolates. Along with the result shows that pfmdr1 does not

affect any sensitivity and chemosentisisation activity in

both wild type and mutation strains.

CHAPTER VI


CONCLUSION

The data present in chapter 4 does not support the

previous study. It shows that both of the wild type isolates

and mutant isolates do not create any different on the

innate antiplasmodial activity and sensitivity of verapamil

in Plasmodium falciparum isolates. Thus, the pfmdr1 does not

mediate any of sensitivity in both wild type and mutant

isolates. Since the p-value of the data is 0.300 by using

the t-test statistical theory, it represents that there is

no significant different between both strains.

Meanwhile, the previous study of Hayward et.al, 2005

explains that there is a significant different between

mutant and wild type isolates means that the pfmdr1 gene is

altering the sensitivity activity in the parasites of both

strains. It also indicated in the previous study that the

introduction of either one or three of the four mutation

isolates in to pfmdr1 gene of sensitive isolates

significantly increased verapamil sensitivity with respect

to the a control transfectant with a wild type pfmdr1 gene.

In this study, it presents an opposed result to the previous

one might because in this study the isolates which were

used, were taking from the nature host, meanwhile, the D10

and 7G8 isolates of previous study were taken from the

clones in the laboratory. In addition, the transfectants


were changed only a gene of pfmdr1 in each isolate but in

this study the isolates do not.

It could be assumed that in this research, the

significant different does not occur because the

transfectants/ isolates might be affected and altered the

sensitivity of verapamil activity by one or more gene, while

in the previous research, the transfectants were infect with

only a gene of pfmdr1. In this study, it is still unknown

whether the transfectants was infected by one or more gene

or not and it could be assumed that there might be others

genes involve in the ability of alteration of isolates since

the isolates were taken from the nature as mentioned before.

The further studies of both isolates gene structures are

necessary.

Furthermore, the data indicated that in the

chemosentisization of chloroquine resistance method, both

wild type and mutant isolates can use the identical fix dose

since there is no significant different between both of

them. It can be assumed that both of them might have the

similar level of sensitivity on the same fix dose.

This result was a primary document for the further

study. Moreover, there are various factors, which need to be

examined such as the accurate mechanism of verapamil to the

isolates and other genes effects. Several repeated

experiment should be continue to make sure that there is no

significant different between and pfmdr1 does not alter both


wild type and mutation isolates on innate antiplasmodial

activity and sensitivity of verapamil in Plasmodium falciparum.

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