In response to growing levels of resistance to currently used antimalarials, there is an urgent need to develop drugs that exhibit novel mechanisms to kill Plasmodium parasites. The objective of this study was to examine the antiparasitic activity of newly synthesized compounds based on imidazolium and triazolium rings. According to our structure/activity relationship studies the key components appear to be their positively charged rings and hydrophobic side groups, and bivalent compounds, which incorporate two positively charged rings, show even greater potency than monovalent compounds. Depending on the concentration used, our compounds appear to primarily inhibit intracellular parasite development or invasion into red blood cells. Selected compounds have been tested in vivo using a P. berghei ANKA murine model. Together, our findings demonstrate that small imidazolium- and triazolium-based compounds display both in vitro and in vivo activity through a novel mechanism of action that may involve inhibition of erythrocyte invasion.

In response to growing levels of resistance to currently used antimalarials, there is an urgent need to develop drugs that exhibit novel mechanisms to kill Plasmodium parasites. The objective of this study was to examine the antiparasitic activity of newly synthesized compounds based on imidazolium and triazolium rings. According to our structure/activity relationship studies the key components appear to be their positively charged rings and hydrophobic side groups, and bivalent compounds, which incorporate two positively charged rings, show even greater potency than monovalent compounds. Depending on the concentration used, our compounds appear to primarily inhibit intracellular parasite development or invasion into red blood cells. Selected compounds have been tested in vivo using a P. berghei ANKA murine model. Together, our findings demonstrate that small imidazolium- and triazolium-based compounds display both in vitro and in vivo activity through a novel mechanism of action that may involve inhibition of erythrocyte invasion.

no / Malaria is a mosquito-borne infectious disease caused by the genus Plasmodium. It causes one million deaths per year in African children under the age of 5 years. There is an increasing development of resistance of malarial parasites to chloroquine and other currently used anti-malarial drugs. Some plant products such as the indoloquinoline alkaloid cryptolepine have been shown to have potent activity against P. falciparum in vitro. On account of its toxicity, cryptolepine is not suitable for use as an antimalarial drug but a number of analogues of cryptolepine have been synthesised in an attempt to find compounds that have reduced cytotoxicity and these have been investigated in the present study in human sperm and lymphocytes using the Comet assay. The results suggest that cryptolepine and the analogues cause DNA damage in lymphocytes, but appear to have no effect on human sperm at the assessed doses. In the context of antimalarial drug development, the data suggest that all cryptolepine compounds and in particular 2,7-dibromocryptolepine cause DNA damage and therefore may not be suitable for pre clinical development as antimalarial agents.

no / New antimalarial drugs are urgently needed to combat emerging multidrug resistant strains of malaria parasites. This Highlight focuses on plant-derived natural products that are of interest as potential leads towards new antimalarial drugs including synthetic analogues of natural compounds, with the exception of artemisinin derivatives, which are not included due to limited space. Since effective antimalarial treatment is often unavailable or unaffordable to many of those who need it, there is increasing interest in the development of locally produced herbal medicines; recent progress in this area will also be reviewed in this Highlight.

Early diagnosis and prompt treatment is one of the malaria control strategies used to minimize malaria morbidity and mortality. One of the mechanisms to implement early diagnosis and prompt treatment is community access to diagnostic services and effective antimalarial drugs. However, in Ethiopia the health system is underdeveloped and much of the rural population has limited access to modern health services. Therefore, the Ethiopian government introduced the Health Extension Programme(HEP) which is a community-based health care delivery system aimed at accessing essential health services through its health extension workers (HEWs). Involvement of the HEWs in prescribing and dispensing antimalarial drugs is shown to have improved community access to antimalarial drugs. However, there is insufficient knowledge of HEWs compliance to malaria treatment guidelines and patient adherence of patients treated by HEWs. The objectives of this study has been to describe the HEWs practice in malaria treatment, to evaluate adherence of patients to antimalarial drugs, to explore the factors influencing the HEWs malaria treatment practice and patient adherence, and to develop the guidelines to support the HEWs in malaria treatment practice. A qualitative study design was used to study the HEWs practice in malaria treatment along with patient adherence. Data were collected using in-depth face-to-face interviews, focus group discussion and patient medical record review and were analysed according to Tesch’s steps. The study revealed that the HEWs adequately comply with malaria treatment guidelines during diagnosis of malaria, as well as during the prescribing and dispensing of antimalarial drugs. However, there are some factors influencing the performance of HEWs. These are: shortage of diagnostic kit/RDT, shortage of antimalarial drugs, patient pressure to obtain coartem, work load, and community beliefs with regard to antimalarial drugs effectiveness. This study also revealed that the HEWs follow up after treatment of patients and good community support systems improved patient adherence to antimalarial drug use. Factors negatively influencing patient adherence were identified to include: forgetfulness, fear of shortage of drugs, adverse drug effects, duration of treatment, rapid relief of malaria symptoms and inadequate awareness of the consequence of incomplete dosage. Guidelines were developed to support the HEWs in malaria treatment practice with the aim to improve patient adherence to antimalarial drugs / Health Studies / D. Litt. et Phil. (Health Studies)

<p>This thesis deals with the development of analytical methods for the determination of new antimalarial drugs in biological fluids. The goal was to develop methods that facilitate clinical studies performed in the field, such as capillary blood sampling onto sampling paper.</p><p>Methods for the determination of atovaquone (ATQ) in plasma, whole blood and capillary blood applied onto sampling paper were developed and validated. </p><p>Automated solid-phase extraction (SPE) and liquid chromatography (LC) with UV absorbance detection was used to quantify ATQ. Venous blood contained higher levels of ATQ than capillary blood after a single dose of Malarone (ATQ + proguanil).</p><p>Ion-pairing LC was used to separate amodiaquine (AQ), chloroquine (CQ) and their metabolites on a CN-column. A method for quantification of AQ, CQ and their metabolites in capillary blood applied onto sampling paper was developed and validated. Perchloric acid and acetonitrile were used to facilitate the extraction of the analytes from the sampling paper. The liquid extract was further cleaned by SPE.</p><p>Methods for the determination of piperaquine (PQ) in plasma and whole blood using SPE and LC were developed and validated. Addition of trichloroacetic acid (TCA) to the samples prior to injection into the LC-system significantly enhanced the efficiency for the PQ peak. Serum and whole blood contained higher levels (about 300 nM) of PQ than plasma (about 200 nM) after a single oral dose of 340 mg PQ. This indicates that PQ may be taken up in the leucocytes and thrombocytes.</p>

This thesis deals with the development of analytical methods for the determination of new antimalarial drugs in biological fluids. The goal was to develop methods that facilitate clinical studies performed in the field, such as capillary blood sampling onto sampling paper. Methods for the determination of atovaquone (ATQ) in plasma, whole blood and capillary blood applied onto sampling paper were developed and validated. Automated solid-phase extraction (SPE) and liquid chromatography (LC) with UV absorbance detection was used to quantify ATQ. Venous blood contained higher levels of ATQ than capillary blood after a single dose of Malarone (ATQ + proguanil). Ion-pairing LC was used to separate amodiaquine (AQ), chloroquine (CQ) and their metabolites on a CN-column. A method for quantification of AQ, CQ and their metabolites in capillary blood applied onto sampling paper was developed and validated. Perchloric acid and acetonitrile were used to facilitate the extraction of the analytes from the sampling paper. The liquid extract was further cleaned by SPE. Methods for the determination of piperaquine (PQ) in plasma and whole blood using SPE and LC were developed and validated. Addition of trichloroacetic acid (TCA) to the samples prior to injection into the LC-system significantly enhanced the efficiency for the PQ peak. Serum and whole blood contained higher levels (about 300 nM) of PQ than plasma (about 200 nM) after a single oral dose of 340 mg PQ. This indicates that PQ may be taken up in the leucocytes and thrombocytes.

Malaria is a life-threatening vector borne infectious disease caused by protozoan parasites of the genus Plasmodium. More than 100 species of Plasmodium can infect numerous animal species such as reptiles, birds and various mammals. However, human malaria is caused by four Plasmodium species -Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae, and occasionally by the simian malaria parasite, Plasmodium knowlesi. Of these, P. falciparum and P. vivax are the major causative agents and P. falciparum is the most virulent. About 300-500 million malaria infections occur every year leading to over 1-2 million deaths, of which 75% occur in African children of less than 5 years infected with P. falciparum. In spite of major global efforts to eliminate this disease over the past few decades, it continues to persist as a major affliction of human-kind imposing serious health and economic burden, especially to the poor countries. In India, the present scenario is about 2 million malaria positive cases every year, with almost 50% being caused by P. falciparum. Although remarkable attempts have been made over the years to develop vaccines against sexual and asexual stages of malaria parasite, an effective vaccine is still not in sight and remains as a distant goal. Hence, highly potent, less toxic and affordable antimalarial drugs remain as a first line therapy for malaria. Unfortunately, these parasites have been evolving against every known antimalarial drug and many of these drugs have lost their potency due to rapid emergence and spread of drug resistant strains. With development of resistance against frontline antimalarials such as chloroquine and antifolates, artemisinin and its derivatives seem to be the only effective antimalarials. However, recent reports on the possible emergence of artemisinin resistant strains, have led to the implementation of artemisinin-based combination therapies as a strategy to prevent drug resistance. Also, this continuous emergence of drug resistance has necessitated the development of new antimalarial drugs to combat this disease. While, Anopheles mosquitoes transmit parasites that infect humans, monkeys and rodents, Culex and Aedes mosquitoes predominate in the natural transmission to birds, and vectors of reptilian parasites are largely unknown. Of the approximately 400 species of Anopheles throughout the world, about 60 are malaria vectors under natural conditions, and 30 of which are of major importance. Ironically, the strategies implemented for controlling Anopheles, have also been hampered by insecticide resistance and other practical difficulties that exist in the scope of their applicability. In the past few years several milestones have been achieved in parasite genome, transcriptome and proteome studies, which could be exploited for the development of new drugs and drug targets. One such promising target includes the metabolic pathways of the malaria parasite which differ significantly from its human host. This thesis entitled “Unique Features of the Heme-Biosynthetic Pathway in Human Malaria Parasite, Plasmodium falciparum” unravels the unique biochemical features of heme-biosynthetic enzymes of P. falciparum, which have the potential for being drug targets. This pathway was first identified in this laboratory over 15 years ago. In the present study, five of the 7 enzymes of this pathway have been cloned, expressed, properties studied and sites of localization identified. With the knowledge on the first two enzymes coming from earlier studies, it is now possible to depict the unique hybrid pathway for heme biosynthesis in P. falciparum with full experimental validation.

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. 121

This thesis describes bioanalytical methods for measuring melatonin and some anti-infective drugs in biological fluids. Solid-phase extraction (SPE) or protein precipitation was used for enrichment and purification of the analytes and Liquid Chromatography (LC) was used to analyze the samples. Developed methods were validated according to international guidelines. Melatonin is a hormone secreted by the pineal gland with a robust circadian rhythm. Bioanalytical methods for determination of melatonin in plasma and saliva have been developed which were used for monitoring melatonin levels in volunteers and patients suffering from sleep related diseases. Eflornithine (DFMO) is a chiral drug used for the treatment of human African trypanosomiasis. A bioanalytical method for determination of the DFMO enantiomers in plasma, after precolumn derivatization with o-phtalaldehyde and N-acetyl-L-cystein has been developed. The method has been used to study the L- and D-DFMO pharmacokinetics, in order to investigate the possible development of an oral treatment of DFMO. A method for simultaneous determination of three antiretroviral drugs i.e. Lamivudine (3TC), Zidovudine (AZT) and Nevirapine (NVP) in dried blood spots (DBS) was developed. The method was used for drug determination in two subjects after receiving standard antiretroviral treatment. The method seemed well suitable for the determination of 3TC and NVP and in some extent for AZT. Lumefantrine (LF) is one of the active components in a new fixed drug combination recommended by the WHO as a replacement to older drugs that has lost their effect. A method for the determination of LF in DBS was developed. The method is suitable for monitoring of drug treatment in rural settings. Tafenoquine is a new promising antimalarial drug under development. A method for the determination of Tafenoquine in plasma and in DBS is described. The method may be useful in future clinical studies in laboratory environment as well as in rural settings. / Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 703