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Role of drug metabolizing enzymes and glutathione system in development of drug resistance in Plasmodium yoelii

IR@CDRI: CSIR-Central Drug Research Institute, Lucknow


Field	Value

Creator	Rizvi, Amber

Date	2014-05-22T06:01:18Z 2014-05-22T06:01:18Z 2010

Identifier	http://hdl.handle.net/123456789/1255

Description	Guide- Dr. Renu Tripathi, PhD. Thesis Submitted to Chhatrapati Shahuji Maharaj University, Kanpur in 2010. “A disaster not just on its way but already happening”. This statement was made by Kevin Marsh in Lancet while talking about malaria. This disease is indeed a disaster that has plagued humanity since ancient times and is still one of the major causes of mortality and morbidity around the world. It is caused by a protozoan Plasmodium, which has a digenetic life cycle. Its asexual stages are propagated inside humans and it results in the pathology associated with the disease. With 1 million deaths and around 250 million cases worldwide that show no signs of abating, malaria control efforts are facing setbacks due to the presence of drug resistant malaria parasites. Antimalarial drug resistance has emerged as one of the greatest challenges facing malaria control today. Drug resistance is cited to be the main cause responsible for the spread of malaria to new areas and re-emergence of malaria in areas where the disease had previously been eradicated. Population movement has resulted in the introduction of resistant parasites to areas that were previously designated free of drug resistance. The economics of developing new pharmaceuticals for tropical diseases, including malaria, demonstrate a huge disparity between the public health importance of the disease and the amount of resources invested in developing new drugs. This disparity coupled with the fact that malaria parasites have currently demonstrated some level of resistance to almost every antimalarial drug currently available, has resulted in a significant increase in the cost incurred for achieving parasitological cure. Drug resistance has rendered the cheap and highly effective chloroquine completely useless along with mefloquine and pyrimethamine-sulphadoxine. Even aretsunate, an artemisinin derivative has reportedly lost its efficacy against Plasmodium falciparum isolated from Pailin in west Cambodia. Various theories have been proposed to explain the phenomenon of multiple drug resistance in malaria. Amongst them is the altered accumulation of drug either due to changes in pH gradient or due to changes in the efflux mechanisms or both; mutations in the genes and changes in drug metabolism. Since the discussion on the mechanism of drug resistance has still not reached a conclusive end, hence it is important to determine the mechanisms involved in drug resistance. More over the information gleaned can then be used to reverse susceptibility of the resistant parasites to the standard anti-malarials. Various models of malaria using non-human malaria parasites have been developed to serve as convenient laboratory references for the provision of biological insight into the human forms of the disease that would otherwise either be practically, or ethically, impossible or difficult to obtain. Animal models of malaria have been widely used since the early 1900s and have facilitated descriptions of the biology of the parasite. Work on such animal models still continues despite the development of in vitro cultivation of P. falciparum by Trager and Jensen, as these investigations lead to the supplementation of information gained through the use of animal models. In some situations the models represent the only feasible avenue of detailed investigation, for example the response of immune system to invasion of hepatocytes by sporozoites or the development of the malaria zygote to the ookinete. Successful and widely used models include a wide range of species that infect laboratory rodents (notably Plasmodium yoelii, Plasmodium berghei and Plasmodium chabaudi), birds (Plasmodium gallinaceum) and primates (notably Plasmodium knowlesi and Plasmodium cynomolgi and, less frequently, Plasmodium reichenowi). All of these species reproduce many of the biological characteristics of human malaria and, this is largely due to the fact that the genus Plasmodium appears to be relatively well conserved at the level of genome organization and individual gene content and structure. Thus rodent malaria parasites serve as a good model for studying drug resistance mechanisms. In order to study the mechanisms of drug resistance in malaria, two rodent strains with differing sensitivities were employed. These strains were P. y. nig. MDR and P. berghei ANKA. The first step was to determine their course of infection inside their host, Swiss mice. The two strains showed different pattern of growth. At the same inoculum P. y. nig. MDR had a much faster rate of multiplication in the host RBCs than P. berghei ANKA. Moreover P. y. nig. MDR is always fatal while in case of P. berghei ANKA, if the inoculum is low enough then after an initial increase of upto 25-30% in parasitaemia, a dip is observed in parasitaemia, with resulting anaemia induced death in the host. After determination of the course of infection it is essential to know the drug sensitivity of the two strains to standard anti-malarials. Thus drug sensitivity tests were performed on the two strains. It was found that P. y. nig. MDR is resistant to CQ at 250mg/kg given for 4 consecutive days, while in case of P. berghei ANKA 16mg/kg of CQ for 4 days cured the infection completely. Thus P. y. nig. MDR is resistant to more than 15 times the dose of CQ required to clear P. berghei ANKA infection. MFQ at 128mg/kg for 4 days does not cure P. y. nig. MDR infection while 16mg/kg for 4 days completely cures P. berghei ANKA infection. For quinine 450mg/kg for 4 days is completely curative for P. y. nig. MDR infection but toxic, in Swiss mice, while for P. berghei ANKA infection 300mg/kg for 4 days is curative. Thus in case of quinine, P. y. nig. MDR needs 1.5 times more drug than that which is required by P. berghei ANKA for completely removing the infection. Since the objective behind the study was to determine the role played by drug metabolizing enzymes, various biochemical assays were done. The assays were of different drug metabolizing enzymes which included cytochrome P450, aniline hydroxylase, aminopyrine-N-demethylase and glutathione-S-transferase. For the role of glutathione system in drug resistance glutathione reductase enzyme was chosen for the study. The major phase I drug metabolizing enzyme Cytochrome P450 (CYP) is present in all the tissues of the body. However it is found in maximum amount in liver, which functions as the major drug and xenobiotics metabolizing organ for the living organisms. CYP was determined in both infected mice livers and in the two rodent strain parasites. The level of CYP in healthy mice livers was found to be 0.6nmol/mg of protein. During the course of infection in Swiss mice through both the rodent strains, the level of enzyme declines perceptibly as the infection level increases. In case of P. berghei ANKA at the highest recorded parasitaemia CYP level was 0.077nmol/mg of protein while in case of P. y. nig. MDR it was 0.062nmol/mg of protein. The decrease seen here is due to the destruction of hepatocytes, which is caused by the multiplying parasites of either strain. When CYP was measured in both the parasites, it was found that the multi drug resistant parasite P. y. nig. had 1.7 times higher amount of CYP than the sensitive strain. This indicates that the high level of CYP has a role to play in drug resistance displayed by the parasites. The second drug metabolizing enzyme studied was aminopyrine-N-demethylase, in infected mice livers and in the two rodent strains. This enzyme belongs to the mono-oxygenase group of phase I drug metabolism, is present in the hepatic microsomes as well. Swiss mice infected with both rodent strains had a decline in the activity of the enzyme, as compared to the enzyme activity present in healthy mice livers. The decrease was sharper in case of mice infected with P. y. nig. MDR, with the group of 50-70% infection showing a decrease of 73.2%, while the same group in case of P. berghei ANKA infection had a decrease of 40.7%. The resistant parasites had a higher activity of the enzyme than the sensitive strain. Aniline Hydroxylase (AH) is another member of phase I group of drug metabolizing enzymes. During infection in Swiss mice of the resistant and the sensitive strain, the activity of AH decreases. Like in case of the other phase I drug metabolizing enzymes covered in this study, this enzyme has a more pronounced decrease with the multi drug resistant strain infection than with the sensitive one. The highest infection group had a decrease of 59.05% with P. y. nig. MDR infection and 45.26% decrease with P. berghei ANKA infection. The resistant parasites had 0.87 nmol/min/mg of protein of AH while the sensitive strain had 0.54 nmol/min/mg of protein of the enzyme. Glutathione-S-Transferase (GST) belongs to phase II of drug metabolism. It neutralizes drugs and xenobiotics to more water soluble compounds and thereby aids in their excretion from the body. Just like the other enzymes, GST also declines in mice livers as the infection increases from either parasite. The decrease for the highest parasitaemia group of 50-70% was 47.5% for P. y. nig. MDR infection while for P. berghei ANKA infection it was just 30.1 %. When GST activity was measured in the two parasite strains, resistant parasites had a higher activity than the sensitive one. Thus drug metabolism plays a very important role in resistance observed in malaria parasites by hampering the elimination of drugs. For determining the role played by the glutathione system, enzyme glutathione reductase (GR) was studied. GR is a component of the glutathione antioxidant system, whose function is to reduce glutathione disulphide in order to maintain the reducing environment of the cell. This enzyme had a direct relationship with infection. As the infection increased so did the activity of GR, in order to protect the cells from the harmful effects of reactive oxygen species formed during the course of infection. The increase was 98.9% for P. berghei ANKA while for P. y. nig. MDR it was a whooping 211% in the highest parasitaemia group of 50-70%. The enzyme was detected in both the strains with the resistant strain showing less activity than the sensitive one. Thus the glutathione antioxidant system also plays a part in the drug resistance problem of malaria parasites. The results from the enzyme assays provide an insight wherein phase I and II drug metabolizing enzymes decline in host during malaria infection. Moreover the enzymes of drug metabolism were found to be in higher amounts in the multi- drug resistant parasites compared to the drug sensitive parasites. This knowledge along with the studies of other researchers was utilized to try to change the susceptibility of the multi-drug resistant parasites. For this CYP enzymes were targeted as they are the major phase I drug metabolizing enzymes. Inhibitors of CYP along with a sub-curative dose of an anti- malarial were given to mice infected with P. y. nig. MDR and the effect of the combination were monitored on the parasitaemia levels. Inhibitors belonging to different classes were used during the study. The first inhibitor to be used was ketoconazole. It is an azole antifungal that was used in combination with α/β arteether (AE) for 100% cure against P. y. nig. MDR infection in Swiss mice. The drug concentrations used were 150mg/kg of ketoconazole and 12.5 and 25 mg/kg of AE (sub-curative dose). The level of CYP was measured in healthy swiss mice livers, dosed with the successful combination as well as in P. y. nig. MDR parasites exposed to the combination. The results demonstrated that ketoconazole inhibited CYP levels in both the mice livers as well as in the parasites, while AE caused an increase in the level which was more pronounced in case of the parasites. The second inhibitor used was fluconazole. It belongs to bis-triazole class of antifungals and was given in combination with mefloquine (MFQ) to mice infected with P. y. nig. MDR and it failed to clear the drug resistant parasites from the host. This was due to the fact that the CYPs involved in MFQ metabolism (CYP3A4) and fluconazole inhibition (CYP2C9) were different. Another antifungal used in inhibitor studies was clotrimazole. It is an imidazole antifungal agent which was used in combination with both chloroquine (CQ) and MFQ. The combination failed to clear the multi drug resistant parasites of P. y. nig. due to the difference in the CYPs being metabolised and inhibited. Clotrimazole inhibited CYP3A1 and CYP3A2 while the CYPs involved in CQ and MFQ metabolism were CYP3A4, CYP2C8 and CYP2D6. Clarithromycin was also used to reverse the susceptibility of anti-malarials in combination against P. y. nig. MDR infection. It is a macrolide antimicrobial agent that was used in combination with AE and CQ. The combination had a 100% cure rate when clarithromycin (150mg/kg, oral) was used with AE (1.25mg/kg, i.m.) and CQ (64 mg/kg, oral). When different route of AE (25 mg/kg oral) was given with clarithromycin (150mg/kg, oral) and CQ (64 mg/kg, oral) the same result of 100% cure was achieved.

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Language	en

Relation	CSIR-CDRI Thesis No. - R-53

Subject	Metabolizing enzymes Glutathione Plasmodium yoelii

Title	Role of drug metabolizing enzymes and glutathione system in development of drug resistance in Plasmodium yoelii

Type	Thesis