Identification Of Chloroquine Resistant Haplotypes Of Plasmodium Falciparum In India And Development Of New Antimalarial Combinations

Abstract

Malaria afflicts 300-500 million people in the world and the mortality ranges from 1-2 million, children in Africa being the most susceptible. With a vaccine not being available against malaria and the front line drugs such as chloroquine and antifolates registering widespread parasite resistance, the challenge of malaria treatment is a formidable task. While, research to discover new drugs has become essential, it has also become necessary to identify therapeutic strategies in the short-term. One approach is to examine whether known drugs used for other applications can be used to treat malaria. A second strategy is to look for natural compounds for antimalarial activity either singly or in combination. Combination therapy has assumed considerable importance in the context of artemisinin derivatives being the sole, tested, efficacious antimalarials left in the basket. A combination therapy with artemisinin derivative may prevent recrudescence due to monotherapy, extend the life of the drug and perhaps bring down the cost of therapy as well. A primary requirement to embark on such studies is to assess the status of drug resistance to the front line drugs in use.

In India, chloroquine is still used as the front line drug for malaria therapy. Although, there have been indications and sporadic reports on the development of chloroquine resistance in the country, there has not been a detailed molecular or clinical evaluation for resistance.

Keeping all these considerations in mind, the objectives of the present study are as follows: 1. Evaluation of chloroquine resistance inP.falciparum isolates from patients using Pfcrt-mutation as marker. 2. Evaluation of the anti-tubercular drugs, rifampicin and isonicotinic acid hydrazide (INH) for antimalarial activity. 3. Evaluation of curcumin from turmeric singly and in combination with α,β- arteether for antimalarial acitivity.

Chapter I deals with the review of literature pertaining to scenario of available antimalarials, efforts to discover new antimalarials based on new drug targets, mechanisms of drug resistance and strategies for combination therapies.

Chapter II deals with an evaluation of Pfcrt mutation in clinical samples of P.falciparum malaria in India. After several false starts to find molecular markers to identify chloroquine resistance, mutations in the Pfcrt gene of P.falciparum, K76T mutation in particular, has been shown to correlate very well with chloroquine resistance in culture. A study of 109 P.falciparum – infected blood samples from different parts of India has revealed that close to 95% of the isolates carry the K76T mutation. This was shown on the basis of susceptibility to ApoI restriction digestion of the PCR product covering this region (264 nt) and DNA sequencing of the PCR product. Interestingly, the resistant haplotype in this region of 72-76 amino acids was found to be mostly SVMNT, except for 4 samples with CVIET haplotype. SVMNT has all along been considered to be of South American origin, where as CVIET is of South East Asian/African origin. Subsequent studies by another group in the country has also shown that the Pfcrt - K76T mutation is seen at least in 85% of the cases and in addition to the dominant SVMNT haplotype, newer haplotypes are also seen. The present study has also included an analysis of N86Y mutation in the Pfmdr1 gene based on susceptibility to Afl III restriction enzyme digestion and DNA sequencing of the PCR product (603 nt). Pfmdr1 mutations have been extensively studied in literature for possible correlation to CQR. The net conclusion is that it does not contribute directly to CQR but may have an indirect correlation. It has been shown in Mali that there is very good correlation between Pfcrt - K76T mutation and Pfmdr1 - N86Y mutation in the P.falciparum isolates. However, in the present study with Indian isolates only around 30% of the samples were found to carry the Pfmdr1 - N86Y mutation. While, further studies on the clinical relevance of the extensive Pfcrt mutation seen in the Indian isolates are needed, it is clear that the genetic change towards chloroquine resistance has already taken place in the Indian context.

Chapter III is devoted to a study of the antimalarial effects of the anti-tubercular drugs, rifampicin and INH. This is on the basis that rifampicin is an inhibitor of prokaryotic and mitochondrial/chloroplast RNA polymerase. P.falciparum harbors the apicoplast, a remnant of chloroplast with a 35kb DNA. It is known that the β, β’- subunits of the apicoplast RNA polymerase are coded by the apicoplast DNA. There is a report that rifampicin is a slow acting antimalarial in cases of P.vivax -nfection. INH is known to act by inhibiting the enoyl-ACP reductase and β - hydroxy ACP synthase in M.tuberculosis. While, M.tuberculosis is known to manifest Fab I and Fab II pathways of fatty acid biosynthesis, it has recently been shown that P.falciparum manifests the FabII (discrete enzymes) pathway. Thus, it was considered possible that INH may also inhibit the fatty acid biosynthetic pathway of P.falciparum leading to inhibition of phospohlipid and membrane biosynthesis.

Studies were, therefore, carried out with rifampicin, INH and the combination on the survival of P.falciparum in culture and P.berghei in mice. With P.falciparum, growth was followed by measuring3[H]-Hypoxanthine incorporation and slide detection of parasites using Giemsa stain. The results indicate that while, rifampicin inhibits P.falciparum growth with an IC50 around 25nM, and INH fails to show any effect even at 200µM concentration. The combination of rifampicin (25nM) and INH (100µM) shows enhanced killing effect. In view of these results, studies were undertaken in mice infected with P.berghei. After 72 hr infection, the mice were orally fed with rifampicin (500 µg/40 g body weight) or INH (1 mg/40 g body weight) or a combination of the two orally for 5 days, starting on day 3. Apart from parasite clearance in blood, protection against mortality is a good index, since all the infected mice die in about 7-8 days. The results indicate that rifampicin leads to around 50% protection and INH treatment gives around 10% protection. However, the combination gives around 83% protection with complete clearance of the parasite in blood. Short- term treatment of infected mice with drugs and an assay of rpoB/C transcription in the parasite using appropriate PCR primers reveal a striking inhibition in combination treatment. Again, when such parasites were put into short-term culture and32P- incorporation into phospholipids was measured, there was striking inhibition with combination treatment. Thus, the results indicate that a combination of rifampicin and INH has potent antimalarial activity in P.berghei-infected mice. The results are dramatic in this case when compared to the results obtained with P.falciparum culture. It is not clear whether the differences are due to differences in action in vitro vs in vivo or due to differences in susceptibility between P.falciparum and P. berghei to the treatment provided.

Chapter IV deals with the antimalarial activity of curcumin (diferuloyl methane) from turmeric singly or in combination with artemesinin or its derivative. Curcumin is reported to have a wide variety of biochemical effects and its anti-cancer activity is under serious investigation. There is an earlier report that curcumin shows antimalarial activity against chloroquine-sensitive P.falciparum. In the present study, curcumin was tested against a chloroquine-resistant culture of P.facliparum and it inhibits growth with an IC50 of 5-8 µM. When P.berghei-infected mice were orally fed with curcumin for 5 days, there was delay in the development of parasitemia, with about 30% of the animals protected against mortality by day 28. For reasons mentioned earlier curcumin was tested in combination with artemisinin/derivative in P.falciparum culture and P.berghei in mice. The results indicate that artemisinin and curcumin have an additive inhibitory effect on P.falciparum growth, based on a detailed analysis of the isobolograms. In terms of the mechanism of action, curcumin treatment leads to accumulation of45Ca in the parasite cytoplasm. It also has a striking inhibitory effect on32P-incorporation into parasite proteins and phospholipids, suggesting an interference with phosphorylation mechanisms. None of these effects are seen under artemisinin treatment, which has been reported to specifically inhibit PfATP6 (Ca ATPase) in P.falciparum. In view of the possible different modes of action of artemisinin and curcumin, the combination was tested in P.berghei-infected mice. The infected mice received a single injection of α,β-arteether and 3 oral doses of curcumin (5mg/30g body weight). Curcumin treatment was found to dramatically delay the onset of parasitemia seen in animals treated with α,β-arteether alone due to recrudescence. In particular, a combination with a single injection of α,β-arteether (750µg or 1.5mg/30g body weight) followed by 3 oral doses of curcumin leads to complete prevention of recrudescence and 100% protection against mortality.

Several combinations with artemisinin derivative are under investigation and they all suffer from toxic side effects, pharmacokinetic mismatch, known resistance to the combining partner and high cost. It is felt that this artemisinin derivative curcumin combination could prove superior in view of the fact that no resistance is known to curcumin and is safe even at very high doses used in the human. Both the drugs are eliminated fast and curcumin is a cheap chemical and available in plenty from natural source (turmeric). In view of these positive attributes, a clinical trial with this combination is recommended.

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