Sensitive and Chloroquine-Resistant Isolates of Plasmodiumfalciparum

Indian Jou rn al of Experimental Biology Vol. 38, November 2000, pp. 1129-1 133

In vitro schizontocidal activity of standard antimalarial drugs on chloroquine­ sensitive and chloroquine-resistant isolates of Plasmodiumfalciparum

Poonam Sharma, C R Pillai* & Jayashri Devi Sharmat School of Environmental Sciences, Jawaharlal Nehru University, New Delhi II 0067, India E mai l: [email protected] *Malaria Research Centre (ICMR), 2 Nanak Encla ve, Delhi II 0009, India

Received 8 Februmy 2000, Revised 2 August 2000

The expanding foci of multiple drug resistant malaria and emergence of different strains requires the reassessment of antimalari al activit y with various drugs. In vitro response of a chl oroquine sensiti ve and a chloroquine resistant isolate of P. falciparum to a group of 6 quinine derived and 3 artemisinin derived standard drugs has been screened, to evalu ate schi zontocidal aciti vt iy of the drugs. In a conventi onal test system the IC511s were derived from the log dose response curves and evalu ated by a ri gorous statistical interpretati on. Analysis by Tukey's test was signifi cant for the quinine related drugs (Q:s;O .O I) and excludes the stati sti cal significance of artemisinin related drugs in these isolates. The dose-responses of these two isolates vary with quinine derivatives, with some overlap at lower doses for the sensiti ve isolate than for the resistant one which manifests at higher doses.

4 In 1820 when th e structure of the principle alkaloid in a reaction catalysed by iron . Artemisinin quinine from Cinchona bark was identified, a large derivatives depend on many mechanism-based 5 number of its synthetic derivatives have been known approaches , and development of endoperoxide 6 to have effects singly and in combination with each antimalarial drugs . other. Since then innumerable attempts at finding the The only reliable definition of resistance in malaria effect of drugs from two major sources quinine and parasites is based on clinical and parasitological artemisinin have been undertaken for the di scovery of response in symptomatic patients and the in vitro their newer formulations and for the understanding of assay. It has the advantage of excluding several host their effect both, in vitro and their pharmacokineti cs factors when good correlations between in vivo and in 7 in clinical trial s. vitro tests can be found . Good correlation between in There is a specific interaction between the parasiti c vivo and in vitro resistance with chloroquine, 8 vesicles and antimalarial agents such as chloroquine amodi aquine and quinine have been reported . and the ability of chl oroquine to raise intra vesicular Mutations are also held responsibl e for chloroquine pH in the parasite at concentrations I-III 00 or less is resistance in Plasmodium falciparum and greatly based on the non-weak base effect. The other theories reduces the sensi ti vi ty to different classes of drugs of chloroquine action are the binding of chloroquine and increases the susceptibility to others like 9 to ferriprotophyrin IX (FP) and the intercalation of aminoquinolines . 1 chl oroquine to DNA • Quinine, mefloquine and The present study has been undertaken to evalu ate halofantrine show subtle deviations from this mode of the schizontocidal effects of various well known 2 action and do inhibit hemozoin product ion . antimalarials and the stati sticall y sign ificant The ethyl ether ex tracti on from Artemisia annua differences in the responses of two isolates of during 1971 and the use of q inghaosu in China Plasmodium falciparum, isolated from clinical cases showed excell ent parasiticidal activit/. The killing of of malaria as prevalent in India. parasi tes by artemi si nin and it s derivatives depends Materials and Methods on a selecti ve enhanced uptake of the drug in specific Quinine, quinaldine, quinacrine, chl oroquine, parasite membranes and itself becoming a free radical amodiaquine, halofantrine, artemisinin, artemether, arteether, primaquine and metronidazole are Sigma *Correspondent author chemicals. Malari a Research Centre P. falciparum 20 1130 INDIAN J EXP BIOL, NOVEMBER 2000

and MRC P. falciparum 76 were chloroquine The number of schi zonts with three or more nuclei sensiti ve and chloroquine resistant isolates of P. (merozoites) per 200 parasites were noted falciparum respectively, used. They were grown and microscopically. The values were compared between maintained in vitro by the standard method 10 and the control and test wells. The percent inhibition of sensitivity to standard drugs was tested foll owing schizont maturati on for each concentration of drug 1 12 star\dard methods 1. • The cultures of th ese isolates was calculated as: I 00 -a, where 'a' is the percent of were synchronized separately using Sorbitol schizont in the test wells as determined by a=zim, treatment and by centrifugation at 2000 rpm for 5 where z and m are mean number of schizonts per 200 min. at room temperature . Before centrifuging them, asexual parasites in test wel ls and control wells they were kept for I 0 min. at room temperature. This respectIv. e I y 14 . ensured killing of all other stages except young rings. From dose-response curves IC50 values (cone. at The supernatant was di scarded and th e pe ll et was which inhibition of parasite growth represent 50%) washed twice using incomplete (serum free) RPMI- were derived for each extract by plotting % inhibition 1640 medium i.e. centrifuged at 2000 rpm for 5 min. again st log concentration. Parasitaemia was adjusted to I- I .5 % for the assay by diluting with human fresh erythrocytes. The cell s Metronidazole, the standard antiamoebic drug was were diluted with compl ete medium to make 8 % al so tested in the same dose range (0.04-0.0 125 haematocrit. Individual drugs (I mg) were di ssolved 11 glml) for both the isolates of P. falciparum. in 50 111 DMSO, 50 111 ethyl alcohol and 900 111 RPMI- I 640 to give rise to I mg/ml concentration. Results These soluti ons were seri all y diluted two fold to The anti pl asmodial activity of the standard drugs in obtain a concentration of I !lglml which were micrograms/ml is shown in Tables I and 2 for both considered as the stock solution. The concentrati on of the isolates, MRC P. falciparum 20 and MRC P. ethanol was 0.05 % in the fin al stock solutions which falciparum 76 respectively. The quinine related was harmless to parasites. For determination of compounds show a fairly graded variation (F-value) intrinsic antimalarial activity, six 2- fold dilutions with lower doses in chloroquine sensitive isolate were prepared (0.4- 0.0 I 25 11 g/ml) to give MRC P . .falciparum 20 (Table I). In the chl oroquine concentration around the range of 50 % inhibitio n resistant isolate, MRC P. falciparum 76 the vari ati ons

(lC50) for all the standard drugs used. During test, 50 are not so significant (Table 2). This is re fl ected in 111 culture medium (with or without drug) were the IC50 values which range I 0 times in chloroquine di spersed into a 96-well microtitre pl ate and next related drugs in CQ sensitive MRC P. falciparum 20 added 50!-ll of infected blood containing an initial and about three times more in CQ resistant MRC P. 1 parasitaemia of 1-1.5% and 8% haematocrit l. All .falciparum 76 (Table 4). The IC50 values with tests were done in triplicate. Artemisinin related drugs show a better value of 17 times in MRC P. falciparum 20, the sensitive isolate The plates were put in the candle j ar and a candl e and a lower effecti veness of three times less with the was lit. When the candle was at the point of resistant isolate MRC P. falciparum 76. The effect of extinction the stopcock was c losed. The candle jar primaquine and metronidazole fa ll s in a range far , was placed inside the incubator for 24-36 hr at 37 .5°C below even the dose-response with that of artemisinin depending upon the development of schi zonts in related drugs (Table 3). Table 4 ranks the IC50 values control wells. After incubation, the contents of the of these drugs with, artemether being the most test we ll s were harvested after removal of the effective and primaquine and metronidazole being the super"natant with a 50!-ll microcapillary tube and the least. Comparisons between the different drugs on the red blood cells deposited at the bottom of the we ll s basis of Tukey's test are given in Table 5. were transferred to a clean microscope slide to form a series of thick films. The resultant thick films were Discussion air-dried for 24 hr and then kept in the incubator set With c hl oroquine related drugs it may be possible at 37.5°C for 30 min, to ensure complete dryness. The to elaborate their mechanism of acti on better in slides were stained for 30 min in Gi emsa stain at a resistant strains than in sensiti ve ones. With dilution of 5-7 % in double di stilled water (v/v). artemi sinin related drugs these studies may be studied SHARMA et al.: DRUG SENSITIVITY IN ISOLATES OF PLASMODIUM FALCIPARUM 11 31 better with sensitive strain s than with the resistant pyronaridine, amodiaquine, chloroquine and qmnme ones. showed positive correlations suggesting cross By the Tukey's test the paired differences of % resistance amongst these drugs m vitro were 15 inhibiton for individual chloroquine related drugs significant at P~ 0.00 1 . which were significant at Q ~ 0.0 I are more The response of these two isolates to artemisinin significant in the sensitive isolate as shown in Tabl e related drugs shows a keener response by an overall 5. In simi lar studies with 161 isolates of P. better ranked schizontocidal activity. The F-values as falciparum from Senegal, the mean IC50 values with derived by ANOVA for chloroquine are not Table !-Inhibition of schizo nt maturati on wi th various chl oroquine rel ated standard dru gs agai nst MRC P. falciparum 20 [Valu es expressed as % inhibition are mean± S E of triplicate observati ons]

Concentrati on (~g/ml) Standard Drug 0.4 0.2 0.1 0.05 0.025 0.0 125

Ch loroquine related dru gs

Quinine 55.80±0.870 49.2±2.943 35.82±2.752 19.63±0.737 14.84±1.369 12.74±2.376 Quinacrine 58.64±0.760 46.77±1.093 34.48±6.280 23 .03±3. 51 8 11.01 ±0.973 5.86±1.917 Quinaldine 60.46±8.34 1 52. 14±7.788 50.46±7 .870 39.53±7.174 28.08±7.211 24.98±7.041 Chloroquine 46.45±7.399 39.64±5.274 33.52±6.050 29.01±3.284 24.44±2.837 6.07±2.001 Halofantrin e 63. 13±2.027 46.97±0.682 35.67±2.955 28.23±0.540 20.06±2.886 12.68±0.597 Amodiaquine 69.25±5.700 46.49±2.263 39.89±0.725 34.64± 1. 11 2 28.75±1.115 20.34±2. 146 F- value 1.60 1 0.989 1.556 4.172* 4.3 14* 5.180*

Artemi sin in 89 .39±0.327 84 .1 7± 1.734 78.29±0.866 75.37±1 .298 65.94±3.854 54.3 7±6.237 Artemether 9 1. 82±1.775 86.48±2.425 82.70±3.863 78. 76±6.075 76.70±7.58 70.54±7.285 Arteether 90.80±1.255 87 .6±2.284 84.96±2.849 74.92±5.820 63.59±2.386 52.99±1 .864 F- va lue 0.927 0.652 1.450 0. 182 1.882 2.995 * p ~ 0.05 F-valu es were derived from one- way ANOV A (inhibition vs . dose).

Table 2-lnhibition of schizont maturation with vari ous chl oroquine related stand ard drugs again st MRC P. falciparum 76 (Valu es expressed as% inhibition are mean ± S E of tri pli cate observati ons)

Stand ard Drug Concentrat ion ( ~ g/ ml ) 0.4 0.2 0. 1 0.05 0.025 0.0 125

Chl oroquine rel ated drugs

Quinine 49.44±6.966 34.64±5. 173 24.21±5.396 14.84±3.98 1 13.28±5.095 9.8 1±3.9 19 Quinacrine 35.36±R.980 27. 88±6.747 22.84±7.064 19.18±6.504 14.14±6.8 15 I I. 14±6.425 Quinald ine 51.3 1±4.728 46.8±6.342 35.52±5.278 3 1.68±3.476 24.2 1±3. 117 17. 15±1.650 Chloroquine 39.46±4.092 30.47±4.779 23.5±4.722 15.78±4.616 12.72±4.550 6.76±2.055 Halofantrine 51.65±0.923 44.20±2. 142 39.86±2.2 12 25 .19±3. 140 18.07±1. 075 I 0.05± 1.536 Amodiaquine 67.46±5.432 61 .35±5.866 53.67±3.366 51.07±3.675 46.78±2.2 19 42.38±2.962 F- valu e 3.199* 2.956 3.092 4.057* 2.481 2.232

Artemi sinin related dru gs

Art emi sinin 77.51 ±0.9 16 73. 13±1.905 69.3±1 .696 61.75±3.416 5 1.63±0.799 35.76±1.2 16 Artemether 85.69±2.82 1 78 .3 4±2.898 72.72±3.827 69. 11 ±4.804 62.60±3.029 58.20±3.183 Arteet her 9 1.46±0.943 84.04±2. ! 84 79.7±2.463 74.72±2.651 67.71±1.695 60.42±2.085 F- va lu e I .756 0.573 0.341 1.051 7.628* 38.5 12 **

P v a lu es*~ 0.05 ; **~0 . 005 F-valu es were derived from one- way A 'OVA (in hibi tion vs. dose). 1132 INDIAN J EXP BIOL, NOVEMBER 2000 significant in MRC P.f20 (Table I) whi le there is a Primaquine, which has an exo-erythrocytic and tendency by MRC P.f 76 to respond better at lower sexual erythrocytic specific activity, shows a truly doses (Table 2) with artemisinin related drugs.

In a study done in Gabon (Nigeria) with 63 Table 4-!C50 (J.Ig/ml) of different standard antimalarial drugs isolates, the 50% inhibitory concentration (IC50) against MRC P.f 20 and MRC P.f 76. They have been ranked in values for artemether were in a narrow range from 0.8 descending order of efficacy and show a consistent pattern for 16 both MRC P.f 20 and MRC P.f 76. Abnormally hi gh values of to 34.8 nM • In thi s study there was a significant quinacrine and metronidazole as seen against, the resistant isolate positive correlation between responses to artemether have resulted, from ex trapolations beyond the actu al dose range and amodiaquine, artemether and chl oroquine , used. artemether and qumme, and artemether and S. No. StandanJ drug ICso (J.Ig/m l) (P halofantrine s; 0.01). Positive correlation between MRC P.J20 MRC P.J76 these drugs suggests in vitro cross-resistance or at Artemether 0.00032 0.0047 least common features in drug uptake and/or mode of 2 Arteether 0.0033 0.007 16 action or resistance . 3 Artemisinin 0.0054 0.026 In the present study the intra-comparisons between 4 Amodiaquine 0.0414 0.159 artemisinin related drugs were not found to be 5 Quinaldine 0.14 1 0.335 significant for MRC P.f 20 at any of the doses 6 Halofantrine 0.214 0.314 employed. Tukey's test for artemisinin related drugs 7 Quinacrine 0.252 4.389 against MRC P.f 76 showed only two combinations 8 Quinine 0.274 0.689 i.e. arternisinin-artemether (-22.44) and artemisinin­ 9 Chloroquine 0.492 1.548 arteether ( -24.66) at 0.0 I 25mg/ml concentration to be 10 Metronidazole 2.403 3 16.967 signi ficant at Qs;O.O I. II Primaquine 4.9 14 4.959

Table 3-lnhibition of schizont maturation with primaquine and metronidazole against MRC P. falcipantm 20 and MRC P. .Jalciparum 76 (Values expressed as% inhibitions are mean± SE of triplicate observations)

Concentration (J.Ig/ml ) Standard Drug 0.4 0.2 0. 1 0.05 0.025 0.0125 Primaquine (MRC Pf 20) 34.39±3.083 30. 16±1.740 24.65±3.600 20.38±1.671 17 .99±2.767 12. 11±5.020 Primaq uine 33. 18±1.775 21.91±3.634 18.02±4.548 13.22±5.928 7.1±2. 122 4.35±2.369 (M RC Pf 76) Metronidazole 35.01±2.789 28.89±4.036 24.68±5.618 19.1 3±3.669 12.86±0.649 5.953±1.936 (M RC Pf 20) Metronidazole 20.03±0.634 18.48± 1.448 15 .74±1.742 11 .47±2.435 I 0.25±2.322 4.76±0.703 (MRC Pf 76)

Table 5-Level of signilicanee Q ~ 0.0 I as derived by Tukey's test for chloroquine related drugs against MRC P.f20 and MRC P.J76. It quantilies the difference between the significant F-values among various drugs at each different concentration. The most signitlcant differences between drugs are found at 0.0125 )lg/ml for the sensitive isolate MRC P.J 20. The resistant isolate shows similar differences between chloroquine- amodiaquine and quinacrine-amodiaquine at a much higher concentration.

Tukey's test for CQ related drugs against MRC P.f20 and MRC P.J76 as mentioned

Comparison, drug-drug Cone. 0.4 J.lg/m l Cone. 0.05J.ig/ml Cone. 0.025J.1g/ml Cone. 0.0 125J.1g/ml Chloroquine- Quinaldine -18.9 1 Chloroquine- Amodiaquine -29.79 (M RC P.f76) - 22.06 -22.34 -36.3 1 Quinacrine-Quinaldine - 17 .07 19.12 Quinac rine-Amodiaquine -33.89 (M RC P.J76) -28.04 -35.77 -36.52 Quinaldine- Quinine 19.9 -36.52 Quinaldine-A modiaquine - 18 .7 - 17 .4 Quinine- Amodiaquine -3 1. 44 -3 1.94 -29.64 - 19.8 (MRC P.J76) Halofantrine- Amodiaquine - 22.84 - 26.72 -29.7 SHARMA eta/. : DRUG SENSITIVITY IN ISOLATES OF PLASMODIUM FALCIPARUM 11 33 different response as compared with the remedy to targeted chemot herapy, Microb Reviews, 60 schizontocidal effect m both these isolates. CQ ( 1996) 30 1. 5 0 ' Neill P M. Bi shop L P, Storr R C, Haw ley S R, Maggs J resistant isolate shows a very high resistance to L, Ward SA & Park B K, Mechani sm- based design or metronidazole. Metronidazole is a broad-spectrum paras ite- targeted artemisinin derivatives: synth esis and antiprotozoal drug, and has been shown to be an an tim alari al ac ti vity of benzylamin o and alk ylamino et her equally strong contender as chloroquine in the cure of analogues of artemisi nin, J Med Chem, 39( 1996) 4511. 17 6 Cumming J N, Pol ypradith P & Posner G H, Antimalari al malaria . acti vity of artemi sini n (quinghaos u) and related tri oxanes: Antiparasitic drugs are seldom completely effective mechanism(s) of ac ti on, Adv Pharmacal, 37( 1997) 253. and complete elimination requires a combination of 7 Rin gwald P & Basco L K, Compari son of in vivo and in vitro drugs, which target parasitic mechanisms, which may tests of resistance in parasites treated with chloroq uin e in be common for various emerging resistant strains in Yaounde, Cameroon, Bull WHO , 77(1999) 34. 8 Segurado A A, di Santi S M & Shiroma M, In vivo and in these protozoa. vitro Plasmodium falciparum resistance to chloroquine, In conclusion, arteether, artemtstmn and amodi aqu ine and quinine in th e Braz ili an Amazo n, Rev l m1 halofantrine show the least difference in resistance in Med Trap Sao Paulo, 39( 1997) 85 . these two isolates. They are better candidates for use 9 Geary T G , Di vo A A & Jensen J B, Activity of quinoli ne­ containing antimalari als again st chloroquine-sensitive and in combination with drugs in the overall response resistant strains of Plasmodium falcipanun in vitro, Trans R. with P. falciparum. . Drug combination studies can Soc Trap Med Hyg .. 8 1(1987) 499. also show synergy between them and attempts to find I 0 Trager W & Jense n J B, Human malaria paras ites in such potentiati on through targeting more than one continuous cultures, Science, 193 ( 1976), 673. effect, in more than one isolate are useful to II Usha Devi C, Pill ai C R, Ad ak T, Sharma V P & Dwi ved i S C, In vitro sensiti vity of Indian isolates of Plasmodium overcome mechanisms of resistance and reversal of falciparum to antimalarial s, J Parasitic Dis, 20 ( 1996) 177. resistance adopted by various plasmodial strains. 12 Ri ec kmann K, Campe ll H G H, Sax L & Mrema J E, Drug sensitivity of Plasmodium falciparum. An in vitro Acknowledgement mi crotechnique, Lancet,! ( 1978) 22. Thanks are due to Dr. Amit Pandey, Malari a Lab., 13 Ang H H, Chan K I & Mak J W, In vilro anti amalari al activity of qu ass inoids from Eurycoma longifolia against International Centre for Genetic Engineering and Mal ays ian chl oroquine-resistant Plasmodium falcipamm Biotechnology (ICGEB), New Delhi for preliminary isolates, Planta Med, 6 1( 1995) 177 . discussions and to Prof. J. 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