Study On Molecular Basis Of Different Reactions Of Plasmodium Falciparum To Various Antimalarial Drugs

Objective To compare the difference of pfcrt and pfmdr1 gene of chloroquine resistant Plasmodium falciparum Fcc SM₁/yN strain at DNA level and RNA expression level after treatment with viarous antimalarial drugs,study molecular biological foundation of different reaction of Fcc SM₁/yN strain to various antimalarial drugs and molecular mechanism involved in the relationship between drug action and resistance for the purpose of providing some helpful clues for development of new drugs that effective to kill drug resistant Plasmodium falciparum strain.Methods(1) Characterization of Fcc SM₁/yN strain by Rieckmann's in vitro micro-test recommended by WHO and PCR-Restriction Fragment Length Polymophism(PCR-RFLP) assay.Determine IC₅₀ value of chloroquine,artemisinin, artemether,daphnetin,chloroquine/ketotifen and chloroquine/cyproheptadin combinations to Fcc SM₁/yN strain respectively and study chloroquine potentiating activity of ketotifen and cyproheptadine in in vitro cultured Fcc SM₁/yN strain by in vitro micro-test.(2) Compare difference of pfcrt gene and pfmdr1 gene of Fcc SM₁/yN strain at DNA level after action of chloroquine,artemisinin,artemether, daphnetin,chloroquine/ketotifen and chloroquine/cyproheptadine combinations respectively by PCR amplification,PCR-Restriction Fragment Length Polymophism (PCR-RFLP) and some other molecular biological methods.(3) Compare difference of pfcrt gene and pfmdr1 gene of Fcc SM₁/yN strain at expression level after treatment of chloroquine,artemisinin,artemether,daphnetin,chloroquine/ketotifen and chloroquine/cyproheptadine combinations respectively by RT-PCR and Real-time PCR methods.Results(1) There are still 4-5 schizonts formed at the well with 32pmol chloroquine and complete schizont inhibition at the well with 64pmol chloroquine.(2) Result of nested PCR-RFLP assay and sequencing indicate the presence of K76T mutation in Fcc SM₁/yN strain,and Fcc SM₁/yN strain carries pfcrt alleles encoding SVMNT haplotype at residudes 72-76.(3) IC₅₀ value of chloroquine,artemisinine,artemether, daphnetin,ketotifen and cyproheptadin to Fcc SM₁/yN strain is 128.95nmol/L(95% confidence interval(CI),119.36-138.53nmol/L),20.16nmol/L(95%CI, 15.67-24.65nmol/L),201.2nmol/L(95%CI,137.46-266.11nmol/L),7.53μmol/L (95%CI,6.84-8.22μmol/L),94.17μmol/L(95%CI,73.32-115.03μmol/L) and 106.74μmol/L(95%CI,82.07-131.41μmol/L) respectively.(4) Ketotifen or cyproheptadin of 0.0098μmol/L-0.078μmol/L had no potentiating effect for chloroquine activity against Fcc SM₁/yN strain.Ketotifen or cyproheptadin of 0.15625μmol/L was found to only potentiate chloroquine of 10-20nmol/L.IC₅₀ was 108.2nmol/L(95%CI,97.48-118.93nmol/L) for chloroquine/ketotifen combination and 113.74nmol/L(95%CI,102.83-124.64nmol/L) for chloroquine/cyproheptadin combination respectively.Activity enhancement index(AEI)([IC₅₀ measured for chloroquine alone-IC₅₀ measured for chloroquine associated with molecules of potentiation]/IC₅₀ measured for chloroquine alone) was 0.16 for chloroquine/ketotifen combination and 0.12 for chloroquine/cyproheptadin combination respectively. Ketotifen or cyproheptadin of 0.3125μmol/L was found to only potentiate chloroquine of 5-20nmol/L.IC₅₀ was 88nmol/L(95%CI,75.76-100.24nmol/L) for chloroquine/ketotifen combination and 97.39nmol/L(95%CI,86.02-108.77nmol/L) for chloroquine/cyproheptadin combination respectively.AEI was 0.32 for chloroquine/ketotifen combination and 0.25 for chloroquine/cyproheptadin combination respectively.Ketotifen or cyproheptadin of 0.625μmol/L was also found to only potentiate chloroquine of 5-20nmol/L.IC₅₀ was 74.53nmol/L(95%CI, 64.8-84.26nmol/L) for chloroquine/ketotifen combination and 89.7nmol/L(95%CI, 77.7-101.7nmol/L) for chloroquine/cyproheptadin combination respectively.AEI was 0.42 for chloroquine/ketotifen combination and 0.3 for chloroquine/cyproheptadin combination respectively.Furthermore,wells added with chloroquine of 5nmol/L combined with ketotifen or cyproheptadin of 0.625μmol/L showed highest degree of decrease in even number of schizonts with 3 or more nuclei among 200 parasites compared to those added only with chloroquine of 5nmol/L(P＜0.05),indicating that this combination had the highest potency of potentiating chloroquine in this study. IC₅₀ value had no obvious difference during 0-7 hours of adding ketotifen or cyproheptadin after chloroquine action.Only a small peak appeared at 6-7 hour with 67.7nmol/L(95%CI,58.02-77.37nmol/L) for chloroquine/ketotifen combination and 81.53nmol/L(95%CI,65.95-97.19nmol/L) for chloroquine/cyproheptadin combination respectively,and AEI was 0.42 for chloroquine/ketotifen combination and 0.3 for chloroquine/cyproheptadin combination respectively.No potentiating activity was found after 8 hours.(5) Result of nested PCR-RFLP assay to detect K76T and N86Y mutations and sequencing of second round PCR products indicate the presence of K76T mutation of pfcrt gene and N86Y mutation of pfmdr1 gene in Fcc SM₁/yN strain before and after treatment of 6 drugs and drug combinations.But the result of nested PCR-RFLP assay to detect D1246Y mutation shows that there are no D1246Y mutation present in Fcc SM₁/yN strain before and after treatment of 6 drugs and drug combinations,with the same phenomenon found in chloroquine sensitive strain Fcc/HN strain set as control.Result of sequencing of second round PCR products also indicates the absence of D1246Y mutation in Fcc SM₁/yN strain before and after treatment of 6 drugs and drug combinations.It is indicated that there is no mutation in site 1246 of pfmdr1 gene and the wild genotype is remained.Results of sequencing of second round PCR products of above three nested PCR amplification and following alignments show that there are no any differences in sequence in these three fragments in Fcc SM₁/yN strain before and after treatment of 6 drugs and drug combinations.(6) There are no any differences in expression levels in both pfcrt and pfmdr1 gene at least within 20 hours of action of these 6 drugs and drug combinations. But it indicates statistically that the expression level of pfcrt gene is different before and after action of chloroquine,artemisinin and chloroquine/ketotifen combination (p＜0.05),and that the expression level of pfmdr1 gene is different before and after action of chloroquine and chloroquine/cyproheptadin combination(p＜0.05).That is the expression level of pfcrt gene increases after action of chloroquine,artemisinin and chloroquine/ketotifen combination,and the expression level of pfmdr1 gene decreases after action of chloroquine and chloroquine/cyproheptadine.All these indicate the tendancies of higher pfcrt gene expression and lower pfmdr1 gene expression after action of chloroquine,artemisinin and chloroquine/ketotifen combination for the former and chloroquine and chloroquine/cyproheptadine for the latter at one IC₅₀ value or one optimal combination dose.RT-PCR indicates the same brightness for all lines compared with the control.Conclusion(1) Fcc SM₁/yN strain is determined to be chloroquine resistant Plasmodium falciparum strain.(2) Appropriate chloroquine/ketotiphen or chloroquine/cyproheptadine combination can potentiate chloroquine against in vitro cultured Fcc SM₁/yN strain.Ketotifen has higher potentiating potency than cyproheptadin at same concentration.0-7 hour during which ketotifen or cyproheptadin is added after chloroquine has no obviously different influence to IC₅₀ value.But no potentiating activity was found after 8 hours.(3) There is no any difference both in pfcrt and pfmdr1 gene at DNA level after treatment of chloroquine, artemisinin,artemether,daphnetin,chloroquine/ketotifen and chloroquine/cyproheptadine combinations of one IC₅₀ value or one optimal combination dose at least within 34 hours of action.(4) There are no any differences in expression levels in both pfcrt and pfmdr1 gene at least within 20 hours of action of these 6 drugs and drug combinations at one IC₅₀ value or one optimal combination dose.But is shows the tendencies of higher pfcrt gene expression and lower pfmdr1 gene expression after action of chloroquine,artemisinin and chloroquine/ketotifen combination for the former and chloroquine and chloroquine/cyproheptadine for the latter at one IC₅₀ value or one optimal combination dose.