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Resistance to artemisinin of malaria parasites (Plasmodium falciparum) infecting alpha thalassemic erythrocytes in vitro Competition in drug accumulation with uninfected erythrocytes

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Resistance to artemisinin of malaria parasites

(Plasmodium falciparum) infecting

alpha-thalassemic erythrocytes in vitro. Competition

in drug accumulation with uninfected

erythrocytes.

S Kamchonwongpaisan, … , M A Avery, Y Yuthavong

J Clin Invest.

1994;

93(2)

:467-473.

https://doi.org/10.1172/JCI116994

.

Plasmodium falciparum infecting hemoglobin (Hb)H and/or Hb Constant Spring

erythrocytes has higher resistance to artemisinin in vitro than when infecting normal

erythrocytes. This is due to low drug accumulation of infected erythrocytes resulting from

competition with uninfected variant erythrocytes, which have a higher accumulation capacity

than genetically normal cells. Drug accumulation of the parasite was shown to be saturable

and dependent on metabolic energy. The 50% inhibitory concentrations (IC50's) for the

parasite in HbH/Hb Constant Spring erythrocytes were decreased when normal

erythrocytes were added to the infected cells, and correspondingly, the IC50's in normal

erythrocytes were increased when HbH/Hb Constant Spring erythrocytes were added to the

infected cells. The changes of IC50 corresponded to the variation in drug accumulation of

mixtures of normal and variant erythrocytes of different compositions. The IC50's for the

parasite in variant erythrocytes were also greatly decreased when the hematocrit of the

culture was lowered, while the IC50's in normal erythrocytes were independent of the

hematocrit. The increase in IC50 values for the parasites infecting variant erythrocytes was

also related to the decrease in parasite accumulation, indicating that drug accumulation

capacity of the parasite also has a role in determining drug sensitivity. Artemisinin sensitivity

therefore is determined by its accessibility to the parasite, which is decreased in infected

variant erythrocytes.

Research Article

Find the latest version:

(2)

Resistance

to

Artemisinin of Malaria

Parasites

(Plasmodium

falciparum)

Infecting

a-Thalassemic

Erythrocytes

In

Vitro

Competition inDrug Accumulation with Uninfected Erythrocytes

SumaleeKamchonwongpaisan,*GanigarChandra-ngam,* Mitchell A.Avery,tandYongyuth Yuthavong *

*Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand, and Departmentof Chemistry, University ofNorthDakota, GrandForks,North Dakota 58202

Abstract

Plasmodium

falciparum infecting hemoglobin (Hb)H

and/or HbConstantSpring erythrocytes has

higher

resistanceto

arte-misinin in vitrothan wheninfecting normal erythrocytes. This is dueto lowdrugaccumulation of infected erythrocytes result-ing from competition with uninfected variant erythrocytes, which have a higher accumulation capacity than genetically

normal cells.Drug accumulation oftheparasitewasshown to

be saturable anddependentonmetabolicenergy. The50% inhib-itoryconcentrations

(IC5,'s)

fortheparasitein

HbH/Hb

Con-stantSpring erythrocytesweredecreasedwhen normal

erythro-cytes were added to theinfected cells,and

correspondingly,

the

IC5R's

innormalerythrocytes wereincreased whenHbH/Hb Constant

Spring

erythrocytes wereaddedtotheinfected cells.

The changesof

IC50

correspondedtothevariationindrug

accu-mulationofmixtures of normal and variant erythrocytes of dif-ferent compositions.The

ICFo's

for theparasiteinvariant

eryth-rocytes werealsogreatly decreased when the hematocrit of the culture was lowered, while the

IC50's

in normal erythrocytes

wereindependentofthehematocrit. The increase in

IC50

values

for the parasites infecting variant erythrocyteswasalso related tothe decrease inparasite accumulation, indicatingthatdrug accumulation capacity oftheparasite also hasarolein deter-mining drug sensitivity. Artemisinin sensitivity thereforeis

de-termined by its accessibilitytotheparasite, which is decreased

ininfectedvarianterythrocytes.(J. Clin.Invest.

1994.93:467-473.) Key words: malaria*Plasmodiumfalciparum*

a-thalas-semia*variant erythrocytes-artemisinin

Introduction

Resistance of the malaria parasite Plasmodium falciparumto

chloroquine

and otherdrugs invariouspartsofthe world

ne-cessitates the development ofneweffective antimalarials

(1).

Derivatives

of artemisinin (qinghaosu)(2, 3)comprise a new

family

of such drugs presently undergoing development and

clinical trials. Their fast actionandeffectivenessagainst

chloro-quine-resistant

strains oftheparasites make them potentially

valuable drugs, especially in such areas of high resistance as

Address correspondence to Dr. Y. Yuthavong, Department of Bio-chemistry, Faculty of Science, Mahidol University, Rama 6 Road,

Bangkok 10400, Thailand.

Receivedforpublication17December1991 and inrevisedform 30

June1993.

Southeast Asia and the Pacific. Theseareasalso have ahigh incidence ofthalassemia and abnormal hemoglobins (4),some

genotypes for which are associated with innate resistance to

falciparum malaria (5, 6). Hence, hemoglobin

(Hb)'H-con-taining erythrocytes from a-thalassemic (genotype

a-thalasse-mia 1 /a-thalassemia 2,

--/

-a)ora-thalassemic/HbConstant

Spring individuals (genotype a-thalassemia 1/Hb Constant Spring,

--/acsa)

have been shown to be resistant to infection by P. falciparum in vitro (7, 8). Enhanced immunerecognition

and clearance of parasitized erythrocytes have also been impli-cated ascontributingtothe apparentprotectiveeffect ofthalas-semia against falciparum malaria (9). Theseexperimental re-sults support an earlier observation that prevalence ofthe genes for a-thalassemia parallels the endemicity of falciparum

ma-laria in the Pacific Islands(5).Thehighfrequenciesof genes for various types of thalassemia and abnormalhemoglobinsin Southeast Asia and various other parts of the world make it

relevant to ask whether antimalarial drugs would have the same

efficacy

in malaria

patients carrying

these genes as in

geneticallynormalpatients. This question also carries implica-tions fordevelopment of drugresistance, whichoriginated in

SoutheastAsia for a number ofantimalarials, including chloro-quine ( 1). If the drug concentrations that are sufficient to kill theparasitesinnormalpatientsweresuboptimalforgenetically variantpatients, these would impose selectivepressure on the

parasitestobecomedrugresistant.

Wehavereportedearlierthat P.falciparum infectingHbH

and/orHb Constant Spring erythrocytes displaysmore resis-tance to artesunate, aderivative of

artemisinin,

and

chloro-quinethan the sameparasitestraininfecting genetically nor-malerythrocytes (10). This novel hosteffectonsensitivityto artesunate isespecially strikingin view of the facts thatthese

genetically variant erythrocytesarealreadysubjecttohigh

oxi-dative stress(11), thatP.

falciparum

issensitive tooxidative

stress( 12-14),andthatartemisinin probablyexertsits action

through

an oxidative mode ( 15-17). Two possible explana-tionsofthe observationwereraised:thehigherlevels ofantioxi-dativeenzymes inthevariant erythrocytes could give riseto a

compensation

effect lessening the oxidativedamagepotential

ofartemisinin, and/or the infected varianterythrocytescould not accumulate as much drug as infected normal erythrocytes. In thispaper, we show that the latter explanation is correct.

Furthermore,we show that the reduction in artemisinin

accu-mulationof infectedvariant erythrocytes is due partly to

com-petition with uninfectederythrocytes forthe drug, and partly to

lowerdrugaccumulation capacities of the parasite.

1. Abbreviations used in thispaper:Hb, hemoglobin;

IC50,

50% inhibi-tory concentration.

ArtemisininResistance ofMalariaParasites ina-ThalassemicErythrocytes 467

J.Clin. Invest.

©TheAmerican Society for Clinical Investigation, Inc.

0021-9738/94/02/0467/07 $2.00

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Methods

CultureofP.falciparum.Chloroquine-resistantK,strainwasobtained

from Kanchanaburi Province, Thailand,andmaintained in vitro by the candle jar technique ofTrager and Jensen ( 18). The cultureswere

synchronizedatringstageby5%sorbitoltreatment(19) and

concen-tratedatschizontstage byPercoll layering technique(20)toobtain 95%parasitemia.The concentratedschizontsweresubculturedwith

normal and variant erythrocytes foratleast 72 hbeforeantimalarial

testingandfor 90 hfordrugaccumulation study,atwhichtimethe synchronywasstilllargelymaintained.Theculturemedium usedwas

RPMI 1640 supplemented with 25 mM Hepes, 0.2% NaHCO3, 40

Ag/ml gentamicin,

and10% humanserum(bloodgroupAB), pH7.4.

50%inhibitoryconcentration(IC50)testing. Theantimalarial activ-ity of artemisinin againstP.falciparum-infectednormal and thalasse-micerythrocyteswasmeasuredbyusingthe [3H]hypoxanthine

incor-poration methodofDesjardins (21).Briefly, artemisininwasdissolved

inDMSOanddilutedwith culture mediumtotheappropriate

concen-tration. Thefinal concentration ofDMSOwas0.001%, which hadno

effectonparasitegrowth.25Alof mediumcontaining drug and 200

Al

of 1.5% cell suspension (or asindicated) with 1-2% parasitemiaat

immature schizont or ring stage (or as indicated) were cultured in

triplicatedmannerfor 24 h. 25 Mlof 0.5 MCi [3H]hypoxanthinewas

added.Afteranadditional18 hofculture,the cellswereharvestedonto

glassfiberfilters. Theradioactivitywasmeasured byaliquid

scintilla-tion counter (LS-1 801; Beckman Instrs., Inc., Fullerton, CA). The

IC50's

(thedrugconcentrations requiredfor 50% reduction of the

radio-activity as compared with control withoutthe drug) ofartemisinin

against theseinfectedcellswereobtainedfromdose-responsecurves.

Insomeexperiments,aconstantamountofP.falciparum-infected

erythrocytes (ring stage) wasdiluted with a known composition of

uninfectednormaland variant erythrocytes, with the final hematocrit of 1.5%. In other experiments, the infected erythrocytes (immature schizontstage)werediluted with variousamountsof normal and

vari-antcellstoobtainasetof cells with various hematocrit values. These

cellsweresimilarlytestedforthe

IC50-["'C]Artemisinin accumulation. After a 90-h culture, P.

falci-parum-infected (mostly at late trophozoite and immature schizont

stage) and controluninfectederythrocytes(both normalandvariant)

werewashedwiththemedium withoutserum supplemented.

Dupli-catedsamplesof 70-Mlpackedcellswereincubated with 630 Mlof 130

nM

15-["'Clartemisinin

(dissolved in 0.001% toluene and 0.1% DMSOinincomplete medium without serum),oratvarious

concen-trations for study of concentrationeffect,at37°Cfor2 h inashaking

waterbath.The cellswerepelleted. 600,lofsupernatantwasbleached

with 400,lof 15% hydrogenperoxideat60°C.4 mlof liquid scintilla-tion fluid wasadded and the radioactivity wasmeasured. Thedrug

accumulated by thecells, definedasdrugaccumulationcapacity,was

calculated and expressedastheamountofdrugin70-Ml pelletperunit

concentration of the drug remainingin thesupernatant(nmol/MM).

Insomeexperiments, drug accumulation byconcentrated infected

erythrocyteswas measureddirectly. Concentrated infected cellswith parasites in latetrophozoite-earlyschizontstagewereprepared.A

syn-chronized culture with 30-40% parasitemiawasput in the medium

supplemented with 2.75,g/mlhypoxanthine.The culturewasthen left understandard incubationcondition for 40h,and theparasitized cells,

mostly at late trophozoite-early schizont stage were harvested and washedwith incompletemedium. 15% cell suspensioninincomplete mediumwasoverlaidon anequal volume of 60% Percolland

centri-fugedat600gfor 20 min. Thedark bandatthe interface of thetwo

media,containing infected cellsat - 95% parasitemia, wascollected

and washedthrice with incomplete medium. Duplicated samplesof 10-Ml packedinfected cell pelletswereused fordeterminingthedrug accumulation asabove. In additionto themeasurement in

superna-tant, thedrug accumulation in the cell pellets was also determined

directly.The pelletswerewashedthrice withincomplete medium, in-cubatedwith 700 Mlof2%SDSsolutionat60'Cfor1h,thenbleached

with 400 ,Al of 15% hydrogen peroxide at 60'C overnight. 4 ml of liquid

scintillation fluidwasaddedand theradioactivitywasdetermined. Inthe study ofeffect of iodoacetamideon drug uptake, the cells were preincubated at370Cwith 500,Ad of100MMiodoacetamide in incomplete medium. After 1 h of incubation, 130 Ml of

['4C]-artemisinin was added to the cell suspension to obtain a final

concen-tration of 100nM and a10% cellsuspension.The cells weremixed and incubated further for 2 h, and the drug accumulation was determined asabove.

Statistical analysis.TheMann-WhitneyUtestwasused for com-paringthedatafromnormalandvariant erythrocytes based on inde-pendentrandom samples.

Results

Ashasbeen observed before, the IC50's forartemisininagainst P.falciparum infecting HbH and HbH/Hb Constant Spring erythrocytes are significantlyhigherthanagainsttheparasitein genetically normal erythrocytes (Fig. 1 ). The basis for the dif-ferences in IC50's wasearlier ( 10) suggested asbeing dueto

differencesinoxidativestresses intheinfected variantand

nor-malerythrocytes, ordueto

differences

in drug accumulation.

The latterpossibility wasexploredthrough theuseof

radiola-beledartemisinin. Fig.2A shows theaccumulation of [

"C]-artemisininatthe initialconcentration of 130nMby infected

genetically normal erythrocytes (6.6% parasitemia), infected HbHerythrocytes(6.6%parasitemia), and HbH/ Hb Constant

Spring

erythrocytes

(5.3%

parasitemia),

while

Fig.

2Bshows

thedrugaccumulation bycontrol erythrocytes thatwere not

exposedtotheparasite. Sincethe final medium concentration

wasdecreasedtodifferentextentsbythe

different

amounts

of

thedrugaccumulated into thecells,the amounts of

accumula-tionweredivided bythe final mediumconcentrationinorder

to obtain normalizedaccumulation, whichcanbe definedas

accumulationcapacities.Fig. 2,DandE,show the accumula-tion per unit medium concentraaccumula-tion ofwhole infected and

con-trolerythrocytes, andFig. 2,CandF, show the difference be-tween the two for genetically normal, HbH, and HbH/Hb

ConstantSpringerythrocytes. The values in Fig.

2,

C andF,

canbe

regarded, respectively,

astheaccumulationand

accu-mulation capacities of the

parasite infecting

the threedifferent

i

0

251

20

15

10-5

0

N HbH Hb H/HbCS

p<0.05 p<O.05

Figure 1. TheIC50values with means±SD for artemisinin

against

P.

falciparum infecting normal(N), HbH, and

HbH/Hb

Constant

Spring(H/CS)erythrocytes.

(4)

(NS) (NS) (p-.004) (p-.001) (p-.004) (p-.001)

1.4

2

w 1.2 0 E

.? 1

Cu

0.

c 0.8

.2

E 0.6

m

8:

c 0.4 E

0

N HbHHbH/CS N HbH HbH/CS (NS) (NS) (p-.004)(p-.001)

N HbH HbH/CS

(p-.004) (p-.001)

Figure 2. The['4C]artemisininaccumulation (A-C) and

accumula-tioncapacity (D-F), with means±SD, by infected erythrocyteswith

6%parasitemia (AandD),uninfectedcontrolerythrocytes (Band

E),and calculated datafor theparasite(C and F). 70 Mul of packed

cellswasincubated with 630Alof medium with initial artemisinin

concentrationof 130 nMfor2 hbefore accumulationmeasurements.

Statisticalanalysis using Mann-Whitney Utestwasshown.N,

nor-mal; HbH/CS, HbH/Hb Constant Spring; NS,notsignificantly dif-ferent.

genetictypesof erythrocytes. While the accumulation and

ac-cumulationcapacities of whole infected(nonparasitizedplus parasitized) cellswerecomparable for the three genetictypes (Fig. 2, A and D), theaccumulationand accumulation capaci-ties for uninfected HbH and, to a greater extent, HbH/Hb ConstantSpring erythrocytesweremuch higher than those of

uninfected normal erythrocytes withvery little accumulation

(Fig. 2, B and E). Bycontrast,theaccumulationand

accumu-lation capacity for the parasite infecting HbH/Hb Constant Spring erythrocytesweresmaller thanthatinfectingHbH

eryth-rocytes,inturnsmaller than that infecting genetically normal

erythrocytes (Fig. 2, C and F).

The accumulation capacities of infected erythrocytes

de-creasedrapidly with increasing initial concentration ofthe drug

upto - 2 MuM for all three genetictypes,indicating saturability

of thebindingsites (Fig. 3). Incontrast,theaccumulation

ca-pacity for uninfected, genetically normal erythrocytes

re-mainedconstantoverthe concentrationrange,whilethe

accu-mulation capacities for uninfected HbH and HbH/Hb

Con-stant

Spring gradually declined

with

increasing

initial

drug

concentration.

The accumulation

capacity

of theparasites

in-fecting

genetically

normal

erythrocytes,

as obtained

by

sub-tractingthoseofuninfected from infected

erythrocytes (Fig.

3 A), wasabouttwoand three times that of the

parasites

infect-ing HbH

(Fig.

3 B) and

HbH/Hb

Constant

Spring

erythro-cytes

(Fig.

3

C), respectively,

at

therapeutic

concentrations

(<100 nM),

indicating differences

in

availability

of

specific

sites.

The

accumulation capacities of

the

parasites infecting

three

different

genetic

types

oferythrocytes

declinedto

approxi-mately thesamelevelat

concentrations

> 2

,gM,

indicating

the predominance of

nonspecific binding

atthese

high

concentra-tions.

Themeasurementof the drug

accumulation

capacity

of the

parasites from

the

difference

in

accumulation of infected

eryth-rocytes(6-10%

parasitemia)

and

uninfected

erythrocytes

was

validatedbydirect measurement

of

drug

accumulation

by

con-centrated

infected

cells

(95%

parasitemia).

Fig.

3Dshows the results

of

suchmeasurement on a

suspension of concentrated

infected

genetically normal and HbH/Hb Constant

Spring

erythrocytes, whichare

comparable

tothe results

obtained by

the

subtraction

method. These results

indicate further

that the

parasites

in

parasitized

cellsaccount for the

majority

of

drug

accumulation.

Metabolic

energy

requirement

for the

drug

accumulation

of

these cellswasalso

studied

by

examining

the effect of temper-ature orof addition

of iodoacetamide.

Fig.

4 Ashows that the decrease in

incubation

temperature

markedly

decreased the

accumulation

capacity

of both infected normal and infected HbH erythrocytes.

Iodoacetamide

hasa

partial effect

on

drug

accumulation of

the

parasites,

but notof

uninfected

erythro-cytes,for both

genetically

normal and HbH

erythrocytes (Fig.

4B).

When the data from drug

sensitivity

and

accumulation

ex-periments,

whichwere

obtained from

thesameset

of samples,

were combined to explore further the

basis

of

differences

in drug

sensitivities,

an inverse

relationship

was

found

between the final medium

concentration of

the

drug incubated

with

uninfected erythrocytes

of

different

genetic

typesand the

IC50

values for parasites infecting these erythrocytes (Fig. 5 A). This indicates that the increase in observed IC50 values of

parasites

infecting variant erythrocyteswasatleastpartly duetolowered

medium concentration.

Fig. 5 B shows that the IC50 values

were correlatedwiththeaccumulation capacities of uninfected

erythrocytes,

whichcanaccount

for

the lowered

medium

con-centration

andincreased

IC50

valuesfor infected variant

eryth-rocytes. In agreement

with

theresults in

Fig.

2, the

accumula-tion

capacity of

the

parasite,

corrected for lowered

medium

concentration,

wasalso

influenced by

the

genetic

type of the erythrocyte that it has infected.

Fig.

5C showsaninverse

corre-lation

between the

IC50

values and

normalized parasite

drug accumulation

capacities, indicating

that theformer is also in-fluenced bythecapacities fordrugaccumulation ofthe

parasite

insidethevariantcells.

Toprovefurtherthat thedifferences inIC50 valuesfor

para-sitesinfecting various genetictypesoferythrocytes were due to

differentdrugaccessibilities, andtoassess therelative

impor-tanceof

competition

by uninfected cellsandparasitedrug

ac-cumulation capacities, ring-stage-infected erythrocytes were culturedinthe presence of thedrugtogether with mixtures of

uninfected genetically normaland

variant

cellsofknown

com-positions for 40h.TheIC50values, obtained before theparasite

ArtemisininResistanceofMalaria Parasites in a-Thalassemic Erythrocytes 469

D E F

I.

(5)

A

0

_- -u--__ .>_\

0

10-7 10,6

Initial concentration (M)

C

0.

0

---a--0

I .. .. I...1

0.8

i10-7

10-6

Initialconcentration (M)

1.4

-i=.

'i

1.2-0

E 1 0. 1

cl

c 0.8

0

0

:3

E0.6

-a) 0 w

0

*a 0.4

-8)

1~0.2

-6

1.4

-a)

1.2-0

E

CL D

1-:

0.8-.2

co

-7 E

0.6-:'

.r-E 0.4

E

0I

o

0.2-10-5

B

*

-a--A

m

---m~~

-,a

D

A

.-- I.I'l ...,7

10.8 10-7 10-6

Initialconcentration (M)

Figure3. Effect ofconcentrationonthe [ '4C]artemisinin accumulation capacity of infected normal (A), HbH(B), HbH/Hb Constant Spring

(C) erythrocytes,and concentratedinfected normaland HbH/Hb Constant Spring (D) erythrocytes, with 9.8, 6.0, 8.9, and 95% parasitemia, respectively (.), uninfected control erythrocytes (i), and calculated data for the parasites(n).70,l (A-C)or 10 Ml(D)ofpacked cellswere

incubated with 630

Al

(A-C)or600

AI

(D) of medium with varying artemisinin concentrationsat370Cfor 2hbeforemeasurements.(m,*,

and n)Points obtained from concentrationdifferencesinthesupernatants(. inD, those forparasitized normal erythrocytes); (o and a)

ob-tainedfrom directmeasurementinpelletof 95%parasitized normal and HbH/Hb Constant Spring erythrocytes, respectively.

could begina newcycle, should be independent of the

compo-sition of uninfected cells with regard totheir genetic types if theseweredetermined only by the accumulation capacities of

theinfected cells, but would be dependenton the uninfected

cell composition if the accumulation capacitiesarewhollyor

partly determined by competition between infected and unin-fected cells. Data in Fig. 6 show that the IC50 values for both infected genetically normal and HbH/Hb Constant Spring erythrocytes indeed dependonthe composition of uninfected cellswithregardtotheirgenetictypes,sothatthe values of IC50

for both genetictypesconverge asthecompositions of the

un-infected cells approachoneanother. The variation inIC50

val-uesoftheparasiteinfecting bothtypesof cellswasreflected by

the variation in accumulation capacities of uninfected erythro-cytes with different compositions of normal and HbH/Hb Constant Springgenotypes(Fig. 7). It cantherefore be

con-cluded that the IC50 valuesaretoalargeextentinfluenced by drug accumulation of uninfected erythrocytes, thereby lower-ing the medium concentration. This conclusion is also

sup-portedby anothersetofexperiments determining theIC50

val-uesfor infected genetically normal and variant erythrocytesat

differenthematocritvalues in the culture. AsFig. 8 shows,the

IC50 values for infected HbH and HbH/Hb Constant Spring

erythrocytesweredecreasedwithdecreasing hematocrit values, whereas the

IC50

values forinfected genetically normal

erythro-cyteswereindependent of the hematocrit. These experiments

show thatthemajorfactorleadingtoapparentdrugresistance

of theparasite infectingthe varianterythrocytesiscompetition indrug accumulation by uninfected variant erythrocytes.

Discussion

The resistancetoartemisinin of P.falciparum infectingHbH

andHbH/HbConstantSpring erythrocytes (Fig. 1) supports

ourpreviousfindingon similarlyincreased resistanceto arte-sunate (10). In this report, wehave shownfurther that such increased resistance ismainlyduetothe fact that uninfected HbH and HbH/Hb Constant Spring erythrocytes have high accumulationcapacity for artemisinin(Figs.2,B andD,3,and 5,AandB),hence, competingforthedrugwithinfected eryth-rocytes.Therefhre,ahigherinitial concentration of thedrugis required so that the infected erythrocytes can accumulate enough drugto achieve the lethal concentration. Uninfected geneticallynormalerythrocytes,onthe otherhand, havelow drugaccumulationcapacity,anddidnotsignificantly compete

for thedrugwith infectednormal erythrocytes. Insupportof

this explanation, itwas found(Fig. 6)thatadecrease in the

470 S.Kamchonwongpaisan, G.Chandra-ngam, M.A.Avery, andY. Yuthavong

1.4

i5

1.2-.D12 E

X

08

E 068

E 0.64

E

0

.E 0.42

0)

1

lo-,

10-5

lnia10-7 1c0-6

Initial concentration(M)

1.4

i

ie 1.2 -0 E

.5Z

1-CL 0

0.

0

E 0.6

-8

ma I

.r

0.4-E

2

0.2-6

-l 10-5

(6)

A Figure 4. (A) Effectof temper-l a b ature on the [

'4C]

artemisinin

1.4-1

accumulation capacityof

nor-1.2. mal (a) and HbH(b)

erythro-cytes.70 ,ul of

packed

cellswas

0_4:

incubatedwith 630

1ul

of

me-dium with initial artemisinin

0.6 concentration of130±7nM

041

at370C(o)and40C(m)for2

0.2-

h before accumulation

mea-Fl L .

M1_E

_ENJsurements.

I, infected erythro-c P c P cytes; C, uninfected control

B

erythrocytes; P,

calculated data

1.6a-

for

parasites.

Error bars

indi-1.4 caterangesof the results from

threeexperiments. (B)Effect ofiodoacetamide on accumu-lationcapacityof normal(a)

0.8 andHbH

(b) erythrocytes.

70

0.6

MAI

of packed cells was

pre-treated with 500 Ml of 100 iM

0.4l iodoacetamide at370Cfor 1

0.2 h,and 130

Al

of

[14CJ-O., _ _ artemisinin was added to a

C P concentration of 100 nM

be-fore further incubation for 2 h(i).(o) Untreated control. I, infected erythrocytes;C, uninfected

controlerythrocytes;P, calculated dataforparasites.Error bars indi-cateranges of the results fromduplicate samples.

25

-20

-

5-257

20

-

10-

5-A 25

-.

20

-15

-.

0

m

10

5-0

0

0

20 40 60 80 100 120 140

Final medium concentration (nM)

C

proportion of

uninfected

HbH/Hb Constant

Spring

erythro-cytesin the culturecaused adecrease in the

IC50 for

P.

falci-parum

infecting

HbH/Hb

Constant Spring

erythrocytes, and

anincrease in the

proportion of uninfected

erythrocytes caused

acorresponding increase inthe IC50 forthe

parasite infecting

genetically normal erythrocytes. The variation in

IC50

values

wasparalleled by thatindrugaccumulation capacities of eryth-rocytemixtures (Fig. 7).Decreaseof the

hematocrit

value in

the culture with variant erythrocytes, thereby leadingto less

drug accumulation by

uninfected

erythrocytes, also led to a

correspondingdecreasein IC50, whilethehematocrit valuehad

little influenceonIC50of parasites infecting genetically normal erythrocytes (Fig. 8).

Although drug accumulation of

uninfected

HbH and

HbH/Hb Constant Spring erythrocytes was far higher than

uninfected genetically

normal erythrocytes, the

accumulation

of allgenetictypes waslinear with externaldrug

concentration

to> 2

,gM.

By contrast,the accumulation by the parasiteswas

saturable, consistent withapreviousreportin whichthe

accu-mulation of dihydroartemisinin showed saturation at drug

concentrations of> 3.86

1uM

(22). Furthermore,temperature

dependenceandpartial inhibition by iodoacetamide of

para-sitedrugaccumulation(Fig. 4) indicated at least partial

depen-denceonmetabolic energy.Exceptfor dependenceon

tempera-tureof HbH erythrocytes, therewaslittle effect oftemperature oriodoacetamideon drugaccumulation of uninfected erythro-cytes.

B

.

.

0

0 , 0 0 0.6 0.8 1

0 0.2 0.4 0.6 0.8 1

Erythrocyteaccumulationcapacity (nmole/,M)

0 0

0

0

.1I.

0.2 0.4 0.6 0.8 1

Parasiteaccumulationcapacity(nmole/VM)

1.2

Figure5.Correlationbetween the IC50 values and (A) final['4C]artemisininconcentration inthemedium of uninfectederythrocytes,r=0.83;

(B) accumulation capacities of uninfectederythrocytes, r= 0.88; and (C) calculateddatafortheaccumulationcapacities of intracellular P.

falciparum, r=0.68.(.) Normal erythrocytes, (c ) HbH erythrocytes, and (in) HbH/Hb Constant Springerythrocytes.

ArtemisininResistanceofMalariaParasitesina-ThalassemicErythrocytes 471

E .-i

C) Cad

Ck

LZ

3E

C.

eG

9 e

Fu

k

c

15

0 - 10

-e

eC

1

(7)

25 6

.

/

0

50

40

-3

-930

-0

/

0

0

20 40 60 80

%Hb H/HbCSerythrocytes

20

10

-100

Figure 6.The ICse valuesfor artemisinin against P.falciparum in-fectingnormal(oand*) and HbH/Hb Constant Spring (o and .) erythrocytesincultures with variouscompositionofuninfected

nor-mal andHbH/Hb ConstantSpring erythrocytes. Data fortwo

exper-imentsareshown.

0

0

-1

/

7,

0

2

7,

x 0~~ 0

0 0

0 0.3 0.6 0.9 1.2 1.5 1.8

%Hematocnt

Figure 8. TheICsOvalues forartemisinin against P.falciparum in-fectingnormal(oand .),HbH(o),andHbH/Hb ConstantSpring, (m)erythrocytesatvarioushematocrit values. Data fortwo

experi-mentsareshown.

Although the data in Figs. 6 and 8 indicate thatthe

IC50

valuesaremainly determinedby the genetictypesand

concen-trations ofuninfected cells that compete for the drug in the

medium, other data (Fig. 2, Cand F, and 5 C) suggestthat

drug accumulation capacity ofthe parasite infectingthe variant

erythrocytes, i.e., the accumulationcorrected foraltered

me-dium concentration,mayalso be another determining factor,

albeitaless importantone. The low drug accumulation ofthe

parasite infectinggenetically variant erythrocytesmaybe due

tolimitationof transit ofthe drug from erythrocytecytoplasm

totheparasiteor atruedecrease in drug accumulation capacity oftheparasite infectingthe variant cells.

Ourresultsreported herecarryimplications for the

treat-ment of P.falciparum-infected patients with artemisinin or

their derivatives, especially in theareaswith high prevalence of

thalassemic/hemoglobinopathic genes, which are largely the

same asthose withmalaria endemicity (5). Hence, ifthe differ-encesinsensitivity alsoexistatthe clinical level, there will bea

0.4

2

0~~~~~~~~~

E

*' 0.31

0

0. E

C

0 20 40 60 80 100

%Hb H/Hb CSerythrocytes

Figure7. The['4C]artemisinin accumulationcapacitiesof mixtures

ofuninfected normal andHbH/HbConstantSpringerythrocytesas afunctionof the percentage of the latter.

potential problem ofpossibleinduction of resistanceto arte-misinin duetoexposureof the parasite infecting variant

eryth-rocytestosubcurative doses of thedrug.Inaddition, theremay

also be problems related with drug toxicity topatients who carrying the genetically variantgenes. Ithas been shown that hemin formsanadduct withartemisinin, which leadsto oxida-tive damage on membranes (23). In addition, it was also shown that ferrous, ferric, hemoglobin, and hemin enhanced the generation of superoxide and hydrogen peroxide by arte-sunate(24). Variant erythrocytes have increased hemichrome (25), nonheme iron (26). Thesecomponentsand other hemo-globin degradative products containing iron may potentiate

artemisinin-induced oxidative damages to the erythrocytes, leadingtosuch consequencesasdecreased erythrocyte

defor-mability and increased methemoglobin level. Eveninthe ab-senceof these drugs,suchdamageshavealreadybeen foundto be morepronounced inerythrocytes containingunstable Hb

(HbSS,HbEE, and HbAE) thanin normalerythrocytes (16). Therefore, the variationineffective dosage and therapeutic

in-dexof artemisinin fortreatmentof P.falciparuminfection in

thesegroupsofpatients should be investigated.

Acknowledgments

This work received financialsupportfrom United Nations

Develop-mentProgram/World Bank/World Health Organization Special

Pro-grammefor Research andTraininginTropical Diseases,the Rocke-fellerFoundation,and the INSERMNord-SudProgramme.

References

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10. Yuthavong, Y., P.Butthep, A. Bunyaratvej, and S. Fucharoen. 1989. Decreasedsensitivityto artesunate andchloroquine of Plasmodiumfalciparum infectinghemoglobin H and/or hemoglobin Constant Spring erythrocytes. J. Clin.Invest.83:502-505.

11.Brunori, M., G. Falcioni, E. Fioretti, B. Giardina, and G. Rotilio. 1975. Formation of superoxide in the autoxidation of the isolated a and 3 chains of humanhemoglobinanditsinvolvementinhemichromeprecipitation.Eur.J. Biochem. 53:99-104.

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13. Friedman, M. J. 1979. Oxidant damage mediates red cell resistance to malaria. Nature(Lond.).280:245-247.

14. Clark,I.A., and N. H.Hunt. 1983. Evidence for reactive oxygen interme-diatescausinghemolysisandparasite death in malaria. Infect. Immun. 39:1-6.

15. Levander, 0.A., A. L.Ager, J. V. C. Morris, and R. G. May. 1989. Qinghaosu, dietaryvitamin E, selenium, and cod-liver oil: effect on the suscepti-bility ofmice to the malarial parasite Plasmodiumyoeldi.Am.J. Clin. Nutr. 50:346-352.

16.Scott, M. D., S. R. Meshnick, R. A. Williams, D. T.-Y. Chiu, H. C. Pan, B. H.Lubin, and F. A. Kuypers. 1989.Qinghaosu-mediated oxidation in normal andabnormal erythrocytes.J.Lab. Clin.Med. 114:401-406.

17.Krungkrai, S. R., andY.Yuthavong. 1987. The antimalarial actionon

Plasmodiumfalciparumofqinghaosuandartesunatein combination with agents whichmodulate oxidant stress. Trans. R.Soc.Trop.Med.Hyg.81:710-714.

18. Trager, W., and J. B. Jensen. 1976. Human malaria parasites in continu-ous culture. Science(Wash. DC). 193:673-676.

19. Lambros,C.,and J. P.Vanderberg. 1979.Synchronizationof Plasmo-diumfalciparum erythrocyticstages in culture.J.Parasitol. 65:418-420.

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References

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