Magister Pharmaceuticae - MPharm / Plasmodium falciparum malaria continues to be a worldwide health problem, especially in developing countries in Africa and is responsible for over a million fatalities per annum. Chloroquine (CQ) is low-cost, safe and was the mainstay aminoquinoline derived chemotherapeutic agent that has been used for many years against blood-stage malaria. However, today the control of malaria has been complicated by increased resistance of the malaria parasite to existing antimalarial agents such as CQ. The primary cause of resistance is mutation in a putative ATP-powered multidrug efflux pump known as the p-glycoprotein (pGP) pump, and point mutation in P. falciparum CQ resistance transporter (PfCRT) protein. These mutations are responsible for the reduced accumulation of CQ at its primary site of action, the acidic digestive food vacuole of the parasite.To overcome the challenges of CQ resistance in P. falciparum, chemosensitiser offer an attractive approach. Chemosensitisers or reversal agents are structurally diverse molecules that are known to reverse CQ resistance by inhibiting the pGP efflux pump and/or the PfCRT protein associated with CQ export from the digestive vacuole in CQ resistant parasites. Chemosensitisers include the well-studied calcium channel blocker verapamil and antihistaminic agent chlorpheniramine. These drugs have little or no inherent antimalarial activity but have shown to reverse CQ resistance in P. falciparum when co-administered with CQ. Because of the channel blocking abilities of pentacycloundecylamines (PCUs) such as NGP1-01, it is postulated that these agents may act as chemosensitisers and circumvent the resistance of the Plasmodium parasite against CQ. Therefore as a proof of concept we conducted an experiment using CQ co- administered with different concentrations of NGP1-01 to evaluate the ability of NGP1-01 to act as a chemosensitiser.Herein, we report the ability of NGP1-01, the prototype pentacycloundecylamine (PCU), to reverse CQ resistance (> 50 %) and act as a chemosensitiser. NGP1-01 alone exhibited very low intrinsic antimalarial activity against both the resistant and sensitive strain (> 2000 nM), with no toxicity to the parasite detected at 10 µM. A statistically significant (p < 0.05) dose dependent shift was seen in the CQ IC50 values at both 1 µM and 10 µM concentration of co-administeredNGP1-01 against the resistant strain. Based on this finding we set out to synthesise a series of novel agents comprising of a PCU moiety as the reversal agent (RA) conjugated to a CQ-like aminoquinoline (AM) molecule and evaluate the potential of these PCU-AM derivatives as antimalarial- and/or reversed CQ agents. As recently shown by Peyton et al., (2012), the conjugation of a CQ-like molecule with a RA such as the chemosensitiser imipramine and derivatives thereof is a viable strategy to reverse CQ resistance in multidrug-resistant P. falciparum. The novel compounds were obtained by amination and reductive amination reactions. The synthetic procedures involved the conjugation of the Cookson’s diketone with different tethered 4-aminoquinoline moieties to yield the respective carbinolamines and the subsequent imines. This was followed by a transannular cyclisation using sodium cyanoborohydride as reducing agent to yield the desired PCU-AM derivatives. The CQ-like AMderivatives were obtained using a novel microwave (MW) irradiation method. Structure elucidation was done by utilising 1H- and 13C NMR spectroscopy as well as IR absorption spectrophotometry and mass spectrometry. Five PCU-AM reversed CQ derivatives were successfully synthesised and showed significant in vitro antimalarial activity against the CQ sensitive strain (NF54). PCU-AM derivatives 1.1 – 1.4 showed antimalarial IC50 values in the ranges of 3.74 – 17.6 ng/mL and 27.6 – 253.5 ng/mL against the CQ-sensitive (NF54) and CQ-resistant strains (Dd2) of Plasmodium falciparum, respectively. Compound 1.1 presented with the highest antimalarial activity against both strains and was found to be 5 fold more active against the resistant strain than CQ. The reversed CQ approach resulted in improved resistance reversal and a significantly lower concentration PCU was required compared to NGP1-01 and CQ in combination. This may be attributed to the improved ability of compound 1.1 to actively block the pGP pump and/or the increased permeability thereof because of the lipophilic aza-PCU moiety. Compound 1.1 also showed the lowest RMI value confirming that this compound has the best potential to act as a reversed CQ agent in the series. Cytotoxicity IC50 values observed for compounds 1.1 – 1.4 were in the low micromolar concentrations (2.39 – 9.54 µM) indicating selectivity towards P. falciparum (SI = 149 – 2549) and low toxicity compared to the cytotoxic agent emetine (IC50 = 0.061 µM).These results indicate that PCU channel blockers and PCU-AM derived conjugates can be utilised as lead molecules for further optimisation and development to enhance their therapeuticpotential as reversal agents and reversed CQ compounds.

Chloroquine was one of the main malarial treatments until the late 1960s when resistance began to emerge. This antimalarial targets haemozoin formation which causes a cytotoxic accumulation of free haem in the malaria parasite leading to parasite death. This is still one of the most promising pathways for treatment of the most prevalent species of malaria parasite, Plasmodium falciparum to date but, owing to growing resistance to chloroquine and other current antimalarial drugs, there is a dire need for new drugs. One strategy is to investigate non-chloroquine haemozoin inhibitors. High-throughput screening (HTS) was previously used to investigate novel β-haematin (synthetic haemozoin) inhibitors with promising P. falciparum growth inhibition activities. Of the 144 330 compounds screened, two hit compounds were selected for investigation in this project with two different scaffolds, namely benzimidazole and carbazole indole. In order to preselect benzimidazole derivatives for synthesis, Discovery Studio and Pipeline Pilot where used in tandem to enumerate 325 728 in silico compounds. These were filtered according to predicted β-haematin inhibition activities, followed by predicted malaria parasite growth activities using previously developed models based on Bayesian statistics. The predicted active compounds were further subjected to an in silico aqueous solubility model and separated according to predicted solubility values however, only 68 out of the 35 124 active compounds showed moderate solubility whilst the rest were poorly soluble. From this data, eighteen compounds were chosen for synthesis with varying functional groups. Using the same Bayesian models, biological activities for seven fragment compounds derived from the benzimidazole hit compound were predicted. Six out of seven were predicted to be β-haematin inhibitors while five out of seven were predicted active against the malaria parasite growth inhibition model. Similar Bayesian predictions were carried out on the seven proposed carbazole indole compounds with three compounds predicted to be β-haematin inhibitors while six compounds were predicted to be active against the malaria parasite growth inhibition model. The eighteen benzimidazole compounds were synthesized using a two-step synthesis, via a condensation reaction using polyphosphoric acid (PPA), 4-aminobenzoic acid and o-phenylenediamine to form the primary amine benzimidazole intermediate after which ani acylation reaction with the appropriate acid chloride furnished the desired compounds. β-haematin inhibition analysis revealed a 78% hit rate compared to the Bayesian predictions which resulted in a 24-fold enrichment compared to random screening. SAR analysis revealed an activity trend related to the position of substituents on the ring system as follows: para < ortho < meta. The type of ring system was also investigated, with a trend of phenyl < furan < pyrrole < thiophene < pyridyl found. The fragment compounds were either purchased or synthesized via standard acylation conditions using acid chlorides or acetic anhydride with primary amines as before. β-haematin inhibition analysis showed all these compounds to be inactive at the 100 µM cut-off but these compounds were still carried through to the next stage of testing in spite of these results. Molecular docking was carried out on all eighteen benzimidazole compounds in Materials Studio using the (001) and (011) β-haematin crystal faces for adsorption, together with a modified CVFF force-field. This showed a correlation between adsorption energies of the (011) β-haematin crystal face with the experimental β-haematin inhibition values. This indicated that the (011) β-haematin crystal face was the most important for β-haematin inhibition. Analysis of the benzimidazole compounds and their π-π and hydrogen bonding interactions was performed. The number of π-π interactions were found to be important for β-haematin inhibition activity. Both sets of benzimidazole compounds were tested against the NF54 chloroquine sensitive malaria parasite using growth inhibition assays with a 50% hit rate shown for the benzimidazole compounds and a 71% hit rate for the fragment study leading to a 26-fold and 36-fold enrichments compared to random screening. SAR analysis of the benzimidazole compounds revealed a trend for activity in relation to substituent position of para ≈ ortho < meta and a ring system trend of phenyl < pyridyl < thiophene < furan < pyrrole. The benzimidazole compounds were further tested against the chloroquine resistant Dd2 P. falciparum strain which showed that disubstituted compounds were more active against this strain. Cellular haem fractionation studies revealed an increase in free haem and decrease in haemozoin confirming that haemozoin inhibition is the mode of action for the benzimidazole compounds. QSAR analysis of these compounds revealed a correlation between the -Log(P. falciparum IC50) which is also known as pLog(P. falciparum IC50) and 1/βhaematin IC50, number of hydrogen bond donors and molecular depth with 1/β-haematin IC50 the most dominant term. iv The first four carbazole indole compounds were synthesized using a two-step synthesis via deprotonation of carbazole and reaction with epichlorohydrin or 1,3-dibromopropane to furnish the epoxide or alkylbromine intermediates. These intermediates underwent a further SN2 reaction using deprotonated indole to furnish four final compounds. Synthesis of another three derivatives required benzyl protection of 7-hydroxyindole alcohol first, followed by reaction with the epoxide intermediates via an SN2 mechanism to furnish the final three compounds. Analysis using the turbidimetric solubility assay revealed the best aqueous solubility range of this series of compounds to be 10-20 µM (moderately soluble). β-haematin inhibition studies were carried out on this series of compounds with a 100% hit rate found when compared to the Bayesian model data which lead to 30-fold enrichment when compared to random screening. SAR analysis showed an increase in the number of hydroxyl groups led to an increase in β-haematin inhibition activity. Docking studies were performed on these seven compounds and showed that hydrogen bonding played a role in anchoring the molecules in the binding pocket on the crystal surface with increased adsorption energies seen with an increase in the number of hydroxyl groups. Malaria parasite growth inhibition studies showed no compounds to be active against the NF54 and Dd2 strains at the 2 µM cut-off. Cellular haem fractionation studies on the carbazole indole compounds showed that this series of compounds acts via a mechanism that results in inhibition of haemoglobin uptake into the food vacuole and not via haemozoin inhibition.

Malaria is a major health problem, mainly in developing countries, and causes an estimated 1 million deaths per year. Plasmodium falciparum is the major type of human malaria parasite, and causes the most infections and deaths. Malaria drugs, like any other drugs, suffer from possible side effects and the potential for emergence of resistance. Chloroquine, which was a very effective drug, has been used since about 1945, but its use is severely limited by resistance, even though it has mild side effects, and is otherwise very efficacious. Research has shown that there are chloroquine reversal agents, molecules that can reinstate antimalarial activity of chloroquine and chloroquine-like drugs; many such reversal agents are composed of two aromatic groups linked to a hydrogen bond acceptor several bonds away. By linking a chloroquine-like molecule to a reversal agent-like molecule, it was hoped that a hybrid molecule could be made with both antimalarial and reversal agent properties. In the Peyton Lab, such hybrid "Reversed Chloroquine" molecules have been synthesized and shown to have better antimalarial activity than chloroquine against the P. falciparum chloroquine-sensitive strain D6, as well as the P. falciparum chloroquine-resistant strains Dd2 and 7G8. The work reported in this manuscript involves simplifying the reversal agent head group of the Reversed Chloroquine molecules, to a single aromatic ring instead of the two rings groups described by others; this modification retained, or even enhanced, the antimalarial activity of the parent Reversed Chloroquine molecules. Of note was compound PL154, which had IC50 values of 0.3 nM and 0.5 nM against chloroquine-sensitive D6 and chloroquine-resistant Dd2. Compound PL106 was made to increase water solubility (a requirement for bioavailability) of the simplified Reversed Chloroquine molecules. Molecular modifications inherent to PL106 were not very detrimental to the antimalarial activity, and PL106 was found to be orally available in mice infected with P. yoelli, with an ED50 value of about 5.5 mg/kg/d. Varying the linker length between the quinoline ring and the protonatable nitrogen, or between the head group and the protonatable nitrogen, did not have adverse effects on the antimalarial activities of the simplified Reversed Chloroquine molecules, in accord with the trends observed for the original design of Reversed Chloroquine molecules as found from previous studies in the Peyton Lab. The simplified Reversed Chloroquine molecules even tolerated aliphatic head groups (rather than the original design which specified aromatic rings), showing that major modifications could be made on the Reversed Chloroquine molecules without major loss in activity. A bisquinoline compound, PL192, was made that contained secondary nitrogens at position 4 of the quinoline ring (PL192 is a modification of piperaquine, a known antimalarial drug that contains tertiary nitrogens at position 4 of the quinoline ring); this compound was more potent than piperaquine which had an IC50 value of 0.7 nM against CQS D6 and an IC50 of 1.5 nM against CQR Dd2, PL192 had IC50 values of 0.63 nM against chloroquine sensitive D6 and 0.02 nM against chloroquine resistant Dd2. Finally, the mechanism of action of these simplified "Reversed Chloroquines" was evaluated; it was found that the simplified "Reversed Chloroquines" behaved like chloroquine in inhibiting β-hematin formation and in heme binding. However, the simplified "Reversed Chloroquines" were found to inhibit chloroquine transport for chloroquine resistant P. falciparum chloroquine resistance transporter expressed in Xenopus oocytes to a lesser extant than the classical reversal agent verapamil. From these studies it was noted that the simplified "Reversed Chloroquines" may not behave as well as classical reversal agents would in restoring chloroquine efficacy, but they are very potent, and so could be a major step in developing drug candidates against malaria.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Malaria is currently one of the most imperative parasitic diseases of the developing world. Current effective treatment options are limited because of increasing drug resistance, treatment cost effectiveness and treatment availability. Novel drug delivery systems are a new approach for increased efficacy in the treatment of the disease. Pheroid™ technology, a proven drug delivery system, in combination with anti-malarial drugs was evaluated in this study. The aim of this study was to evaluate the possible enhancement of the efficacy of the existing anti-malarial drugs in combination with Pheroid™ technology. The efficacy of existing anti-malarial drugs in combination with Pheroids was investigated in vitro with a chloroquine RB-1-resistant strain of P. falciparum. Two different Pheroid formulations, vesicles and microsponges, were used and the control medium consisted of sterile water for injection. Parasitaemia levels were determined microscopically and expressed as a percentage. An in vivo pilot study was also conducted using the P. berghei mouse model. The mice were grouped into seven batches of three mice each. The control group was treated with a Pheroid vesicle formulation only. Three of the groups were treated with three different concentrations of chloroquine dissolved in water namely 2 mg/kg; 5 mg/kg and 10 mg/kg bodyweight (bw) respectively, while the other three groups received the same three concentrations of chloroquine entrapped in Pheroid vesicle formulations. The measure of parasite growth inhibition (percentage parasitaemia), the survival rates and the percentage chemosuppresion was determined. In the in vivo study, all concentrations of chloroquine entrapped in Pheroid vesicles showed suppressed parasitaemia levels up to 11 days post infection. From day 11, the parasitaemia increases rapidly and becomes higher than that in groups treated with chloroquine in water. Chloroquine entrapped in Pheroid vesicles showed improved activity against a chloroquine resistant strain (RB-1) in vitro. The efficacy was enhanced by 1544.62%. The efficacy of mefloquine, artemether and artesunate in Pheroid microsponges were enhanced by 314.32%, 254.86% and 238.78% respectively. It can be concluded that Pheroid™ technology has potential to enhance the efficacy of anti-malaria drugs. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2008.

Resistance to antimalarial drugs continues to be a major obstacle in controlling and eradicating malaria. The identification of genetic markers of resistance is vital for disease management but they can be difficult to predict before resistance arises in the field. This thesis describes an alternative approach to gene identification, combining an in vivo experimental evolution model, Linkage Group Selection (LGS) and Solexa genome re-sequencing. Here this model was used to resolve the genetic basis of chloroquine and artemisinin resistance in the rodent malaria parasite Plasmodium chabaudi. AS-30CQ is a parasite with high resistance to chloroquine and resistance to artemisinin. It was crossed with the genetically different drug-sensitive strain AJ. The resulting progeny were selected with drugs and backcrossed to the sensitive parent. Both crosses were treated with increasing concentrations of chloroquine and artemisinin. The frequency of markers from the sensitive parasite were analysed in order to characterize the signatures of drug selection. Three loci involved progressively in chloroquine resistance were identified on chromosomes 11, 3 and 2. One main locus on chromosome 2 was identified with artemisinin selection. The Solexa platform was used to re-sequence the genomes of both AS-30CQ and its sensitive progenitor, AS-sens. The differences between the two genomes were integrated with the LGS data to identify: 1) a strong candidate for the main CQresistance determinant - a putative amino acid transporter on chromosome 11 (aat1) 2) two candidates for high level chloroquine resistance on chromosome 3. and 3) a mutation in ubp1 gene on chromosome 2 that is likely to contribute to the highest level of chloroquine resistance and be main determinant of the artemisinin resistance phenotype. In addition the last section of this thesis describes two otherwise isogenic clones showing low- and high levels of chloroquine resistance were grown competitively to evaluate the effect of these mutations on parasite fitness. The highly resistant strain demonstrated a loss of fitness in relation to its more sensitive progenitor and was outcompeted in untreated and low-treated infections.

Malaria is considered as one of the most prevalent and debilitating diseases affecting humans. Plasmodium falciparum is the most virulent form of the parasite which developed resistance to several antimalarial drugs. Chloroquine is one of the most successful antimalarials developed that is safe, effective, and cheap. However, its use has been limited due to the emergence of drug resistance. Click chemistry, particularly, the copper(I)-catalyzed reaction between azides and alkynes has shown to have a cutting-edge advantage in medicinal chemistry by its reliability, selectivity and biocompatibility. Triazole-based antimalarials were synthesized via copper(I)-catalyzed alkyne-azide cycloaddition reaction by modifying the aliphatic chains terminal of chloroquine. The compounds synthesized contain triazole ring directly connected to an aromatic ring or via a piperazine linker. When tested for their in vitro antimalarial activity against D6, Dd2 and 7G8 strains of P. falciparum, 12 out of 28 compounds showed better activity against chloroquine resistant strains. Particularly, PL403 and PL448 exhibited potent activity than chloroquine against CQ-resistant strains Dd2 and 7G8, with IC50 values of 12.8 & 14.5 nM, and 15.2 & 11 nM respectively. The efficiency of synthesizing several triazole-based antimalarials have proven click chemistry to be fast and efficient reaction. Generally, para-substitutions and di-substitutions with electron-withdrawing groups were found to be beneficial for having better antimalarial activity for these group of click compounds. Moreover, the incorporation of piperazine linker has brought an enhanced antimalarial activity.

Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2008.

Porphyria is a rare metabolic disease which occurs as a result of accumulation of endogenous porphyrins due to specific enzyme deficiency in the biosynthetic pathway of heme. Chloroquine is currently used in the treatment of cutaneous porphyria, although its mechanism of action is not yet well understood. It is believed that chloroquine works in porphyria by forming complexes with excess porphyrin molecules and thus enhancing their elimination from the body. Previous reports of porphyrin-chloroquine complexes have been done mostly in aqueous models. In this study, UV/Visible optical absorbance difference spectroscopy was used to study the complexation of protoporphyrin IX with chloroquine and a range of acceptor molecules in hydrophobic models. The results show that chloroquine, mefloquine, amodiaquine, quinacrine, and pyronaridine formed relatively stronger complexes compared to other molecules such as quinine, duroquinone and caffeine. Therefore, relative to chloroquine, some of the molecules with comparable or greater binding affinity to protoporphyrin IX might also be useful in the treatment of porphyria.

Malaria is currently one of the most imperative parasitic diseases of the developing world. Current effective treatment options are limited because of increasing drug resistance, treatment cost effectiveness and treatment availability. Novel drug delivery systems are a new approach for increased efficacy in the treatment of the disease. Pheroid™ technology, a proven drug delivery system, in combination with anti-malarial drugs was evaluated in this study. The aim of this study was to evaluate the possible enhancement of the efficacy of the existing anti-malarial drugs in combination with Pheroid™ technology. The efficacy of existing anti-malarial drugs in combination with Pheroids was investigated in vitro with a chloroquine RB-1-resistant strain of P. falciparum. Two different Pheroid formulations, vesicles and microsponges, were used and the control medium consisted of sterile water for injection. Parasitaemia levels were determined microscopically and expressed as a percentage. An in vivo pilot study was also conducted using the P. berghei mouse model. The mice were grouped into seven batches of three mice each. The control group was treated with a Pheroid vesicle formulation only. Three of the groups were treated with three different concentrations of chloroquine dissolved in water namely 2 mg/kg; 5 mg/kg and 10 mg/kg bodyweight (bw) respectively, while the other three groups received the same three concentrations of chloroquine entrapped in Pheroid vesicle formulations. The measure of parasite growth inhibition (percentage parasitaemia), the survival rates and the percentage chemosuppresion was determined. In the in vivo study, all concentrations of chloroquine entrapped in Pheroid vesicles showed suppressed parasitaemia levels up to 11 days post infection. From day 11, the parasitaemia increases rapidly and becomes higher than that in groups treated with chloroquine in water. Chloroquine entrapped in Pheroid vesicles showed improved activity against a chloroquine resistant strain (RB-1) in vitro. The efficacy was enhanced by 1544.62%. The efficacy of mefloquine, artemether and artesunate in Pheroid microsponges were enhanced by 314.32%, 254.86% and 238.78% respectively. It can be concluded that Pheroid™ technology has potential to enhance the efficacy of anti-malaria drugs. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2008.