Plasmodium knowlesi was initially identified in the 30s as a natural Plasmodium of Macaca fascicularis monkey also capable of experimentally infecting humans. It gained a relative notoriety in the mid-30s as an alternative to Plasmodium vivax in the treatment of the general paralysis of the insane (neurosyphilis). In 1965 the first natural human infection was described in a US military surveyor coming back from the Pahang jungle of the Malaysian peninsula. P. knowlesi was again brought to the attention of the medical community when in 2004, Balbir Singh and his co-workers reported that about 58% of malaria cases observed in the Kapit district of the Malaysian Borneo were actually caused by P. knowlesi. In the following years several reports showed that P. knowlesi is much more widespread than initially thought with cases reported across Southeast Asia. This infection should also be considered in the differential diagnosis of any febrile travellers coming back from a recent travel to forested areas of Southeast Asia. P. knowlesi can cause severe malaria with a rate of 6-9% and with a case fatality rate of 3%. Respiratory distress, acute renal failure, shock and hyperbilirubinemia are the most frequently observed complications of severe P. knowlesi malaria. Chloroquine is considered the treatment of choice of uncomplicated malaria caused by P. knowlesi.
Since 2005, artesunate (AS) plus sulfadoxine/pyrimethamine (SP) combination has been adopted as the first-line treatment for uncomplicated malaria in Yemen in response to the high level of Plasmodium falciparum resistance to chloroquine (CQ). Therefore, the aim of the present study was to determine the frequency distribution of molecular markers associated with resistance to CQ and AS plus SP combination among P. falciparum isolates from a malaria-endemic area in Taiz governorate, Yemen. Fifty P. falciparum isolates were collected during a cross-sectional study in Mawza district, Taiz, in the period from October 2013 to April 2014. The isolates were investigated for drug resistance-associated molecular markers in five genes, including P. falciparum CQ resistance transporter (pfcrt) 76T and P. falciparum multidrug resistance 1 (pfmdr1) 86Y as markers of resistance to CQ, mutations in the Kelch 13 (K13) propeller domain for resistance to AS, and P. falciparum dihydrofolate reductase (pfdhfr) and P. falciparum dihydropteroate synthase (pfdhps) genes for resistance to SP. Nested polymerase chain reaction was used to amplify target genes in DNA extracts of the isolates followed by restriction fragment length polymorphism for detecting 76T and 86Y mutations in pfcrt and pfmdr1, respectively, and by DNA sequencing for detecting mutations in K13, pfdhfr and pfdhps. All the investigated isolates from Mawza district were harboring the pfcrt 76T mutant and the pfmdr1 N86 wild-type alleles. The pfdhfr 51I/108N double mutant allele was found in 2.2% (1/45) of the isolates; however, no mutations were detected at codons 436, 437, 540, 581 and 613 of pfdhps. All P. falciparum isolates that were successfully sequenced (n=47) showed the K13 Y493, R539, I543 and C580 wild-type alleles. In conclusion, the pfcrt 76T mutant allele is fixed in the study area about six years after the official withdrawal of CQ, possibly indicating its over-the-counter availability and continued use as a self-medication in the study area. However, the almost predominant wild-type alleles of the genes associated with resistance to AS and SP among P. falciparum isolates in the present study indicates the sustained efficacy of the currently adopted first-line treatment of AS plus SP in the study area.
Imatinib, a selective inhibitor of c-KIT and Bcr-Abl tyrosine kinases, approved for the treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors, shows further therapeutic potential for gliomas, glioblastoma, renal cell carcinoma, autoimmune nephritis and other neoplasms. It is metabolized by CYP3A4, is highly bound to alpha-1-acid glycoprotein and is a P-glycoprotein substrate limiting its brain distribution. We assess imatinib's protein binding interaction with primaquine, which also binds to alpha-1-acid glycoprotein, and its metabolic interaction with ketoconazole, which is a CYP3A4 inhibitor, on its pharmacokinetics and biodistribution. Male ICR mice, 9-12 weeks old were given imatinib PO (50 mg/kg) alone or co-administered with primaquine (12.5 mg/kg), ketoconazole (50 mg/kg) or both, and imatinib concentration in the plasma, kidney, liver and brain was measured at prescheduled time points by HPLC. Noncompartmental pharmacokinetic parameters were estimated. Primaquine increased 1.6-fold plasma AUC(0)--> infinity, C(Max) decreased 24%, T(Max) halved and t(1/2) and mean residence time were longer. Ketoconazole increased plasma AUC(0)-->infinity 64% and doubled the C(Max), but this dose did not affect t(1/2) or mean residence time. When ketoconazole and primaquine were co-administered, imatinib AUC(0)-->infinity and C(Max) increased 32 and 35%, respectively. Ketoconazole did not change imatinib's distribution efficiency in the liver and kidney, primaquine increased it two-fold and it was larger when both the drugs were co-administered with imatinib. Ketoconazole did not change brain penetration but primaquine increased it approximately three-fold. Ketoconazole and primaquine affect imatinib clearance, bioavailability and distribution pattern, which could improve the treatment of renal and brain tumors, but also increase toxicity. This would warrant hepatic and renal functions monitoring.
Malaysian, TGR (Thailand), and Gambian (West African) Plasmodium falciparum isolates were cultured in vitro by the candle jar method and were characterized for their susceptibilities to present antimalarial drugs by the modified in vitro microtechnique. Results showed that 93 and 47% of the Malaysian isolates were resistant at 50% inhibitory concentrations of 0.1415 to 0.7737 and 0.1025 to 0.1975 microM, respectively, while the rest were susceptible to choloroquine and cycloguanil at 0.0376 and 0.0306 to 0.0954 microM, respectively. All isolates were susceptible to mefloquine, quinine, and pyrimethamine at 0.0026 to 0.0172, 0.0062 to 0.0854, and 0.0149 to 0.0663 microM, respectively. In contrast, the Gambian isolate was susceptible to multiple drugs at 0.0024 to 0.0282 microM; TGR was resistant to chloroquine at 0.8147 microM but was susceptible to mefloquine, quinine, cycloguanil, and pyrimethamine at 0.0024, 0.0096, 0.0143, and 0.0495 microM, respectively.
The emergence and spread of drug-resistant Plasmodium falciparum have been a major impediment for the control of malaria worldwide. Earlier studies have shown that similar to chloroquine (CQ) resistance, high levels of pyrimethamine resistance in P. falciparum originated independently 4 to 5 times globally, including one origin at the Thailand-Cambodia border. In this study we describe the origins and spread of sulfadoxine-resistance-conferring dihydropteroate synthase (dhps) alleles in Thailand. The dhps mutations and flanking microsatellite loci were genotyped for P. falciparum isolates collected from 11 Thai provinces along the Burma, Cambodia, and Malaysia borders. Results indicated that resistant dhps alleles were fixed in Thailand, predominantly being the SGEGA, AGEAA, and SGNGA triple mutants and the AGKAA double mutant (mutated codons are underlined). These alleles had different geographical distributions. The SGEGA alleles were found mostly at the Burma border, while the SGNGA alleles occurred mainly at the Cambodia border and nearby provinces. Microsatellite data suggested that there were two major genetic lineages of the triple mutants in Thailand, one common for SGEGA/SGNGA alleles and another one independent for AGEAA. Importantly, the newly reported SGNGA alleles possibly originated at the Thailand-Cambodia border. All parasites in the Yala province (Malaysia border) had AGKAA alleles with almost identical flanking microsatellites haplotypes. They were also identical at putatively neutral loci on chromosomes 2 and 3, suggesting a clonal nature of the parasite population in Yala. In summary, this study suggests multiple and independent origins of resistant dhps alleles in Thailand.
The development of resistant to current antimalarial drugs is a major challenge in achieving malaria elimination status in many countries. Therefore there is a need for new antimalarial drugs. Medicinal plants have always been the major source for the search of new antimalarial drugs. The aim of this study was to screen selected Malaysian medicinal plants for their antiplasmodial properties.
Studies have shown that the barks and roots of some Apocynaceae species have anticancer and antimalarial properties. In this study, leaf extracts of five selected species of Apocynaceae used in traditional medicine (Alstonia angustiloba, Calotropis gigantea, Dyera costulata, Kopsia fruticosa and Vallaris glabra) were assessed for antiproliferative (APF) and antiplasmodial (APM) activities, and analysed for total alkaloid content (TAC), total phenolic content (TPC) and radical-scavenging activity (RSA). As V. glabra leaf extracts showed wide spectrum APF and APM activities, they were further screened for saponins, tannins, cardenolides and terpenoids.
Exposure of Plasmodium falciparum to increasing sublethal drug concentrations followed by drug treatment led to the development of many resistant parasites. Therefore, the susceptibility of these clones to the type II antifolate drugs, cycloguanil and pyrimethamine, before and after subculturing them in vitro for a period of 3 years, was studied.
Six clones were derived from each Plasmodium falciparum isolate obtained from Malaysia, Africa and Thailand and were characterized against type II antifolate drugs, cycloguanil and pyrimethamine using the modified in vitro microtechnique. Results showed that these isolates were of a heterogeneous population, with 50% inhibitory concentrations of Gombak A clones at 0.0151-0.1450 and 0.0068-0.1158 microM, Gambian clones at 0.0056-0.1792 and 0.0004-0.0068 microM and TGR clones at 0.0103-0.0703 and 0.0776-0.3205 microM against cycloguanil and pyrimethamine, respectively. All clones displayed similar susceptibilities as their parent isolates except A/D3, A/D5, A/G4 and A/H7 clones which were sensitive to cycloguanil at 0.0735, 0.0151, 0.0540 and 0.0254 microM but Gm/B2 clone was resistant at 0.1792 microM, respectively. However, A/D3, TGR/B4, TGR/B7, TGR/C4, TGR/C7 and TGR/H2 clones were resistant to pyrimethamine at 0.1158, 0.1070, 0.1632, 0.1580, 0.2409 and 0.3205 microM, respectively. Further results indicated that they were pure clones compared to their parent isolates as their drug susceptibility studies were statistically different (p < 0.05).
Eleven Malaysian Plasmodium falciparum isolates were cultured in vitro and later subjected to antimalarial evaluations in 96-well microtiter plates. After cryopreservation, the IC50 (nM) for ST 195, ST 196, ST 197, ST 244 and ST 245 isolates were, respectively: 180.9, 198.7, 482.0, 580.0 and 690.1 for chloroquine; 3.4, 3.4, 9.2, 4.0 and 5.8 for mefloquine; 21.9, 10.5, 40.7, 40.1 and 48.7 for quinine; 136.7, 58.8, 116.4, 29.4 and 95.4 for cycloguanil, and 48.3, 57.5, 47.4, 61.5 and 37.8 for pyrimethamine. Before cryopreservation they were 172.5, 141.5, 453.2, 636.0 and 651.6 nM for chloroquine; 4.8, 2.6, 9.0, 6.9 and 5.8 nM for mefloquine; 21.3, 8.3, 41.9, 49.6 and 40.1 nM for quinine, 129.9, 47.3, 109.3, 30.6 and 95.4 nM for cycloguanil, and 45.4, 47.4, 40.2, 66.3 and 36.0 nM for pyrimethamine. IC50 (nM) for Gombak A, Gombak C, ST 9, ST 12, ST 85 and ST 148 isolates after 12 months of continuous in vitro culture were, respectively: 477.0, 492.3, 367.1, 809.4, 566.5 and 341.8 for chloroquine; 2.9, 11.1, 8.5, 16.9, 5.3 and 4.2 for mefloquine; 6.2, 58.3, 52.7, 36.7, 31.8 and 26.2 for quinine; 154.5, 57.2, 130.3, 94.2, 81.4 and 102.9 for cycloguanil, 26.9, 24.9, 43.8, 31.0, 14.1 and 56.7 for pyrimethamine. Before the 12-month culture they were 472.3, 452.9, 352.7, 773.7, 702.7 and 322.7 nM for chloroquine; 2.6, 13.2, 8.5, 17.2, 5.0 and 4.0 nM for mefloquine; 6.2, 85.4, 53.9, 38.5, 35.8 and 38.5 nM for quinine; 106.8, 74.3, 112.4, 89.8, 91.8 and 103.3 nM for cycloguanil, and 26.9, 31.4, 47.0, 28.1, 14.9 and 56.7 nM for pyrimethamine. Thus none of these isolates differed in their original susceptibilities after either of these procedures.
Plasmodium falciparum isolates from Malaysia, Africa and Thailand were cultured in vitro following the method of Trager and Jensen and subsequently cloned using the limiting dilution method of Rosario. These clones were presently characterized against three schizonticidal drugs, chloroquine, mefloquine and quinine, using the modified in vitro microtechnique. Results showed that all the clones derived from Gombak A isolate were chloroquine-resistant with average IC50 values ranging at 0.1377-1.0420 microM (0.007-0.058 mefloquine activity), sensitive to mefloquine at 0.0032-0.0103 microM and quinine at 0.0025-0.0428 microM (0.075-3.080 mefloquine activity). Similarly, the TGR clone displayed resistance to chloroquine at 0.1715-0.5875 microM (0.002-0.029 mefloquine activity) but were also sensitive to mefloquine at 0.0008-0.0058 microM and quinine at 0.0055-0.0700 microM (0.055-0.202 mefloquine activity). In contrast, four out of six Gambian clones were sensitive to chloroquine at 0.0047-0.0172 microM (0.122-0.617 mefloquine activity) but all were sensitive to mefloquine at 0.0008-0.0029 and 0.0016-0.0102 microM (0.096-1.813 mefloquine activity). In general, most of the clones displayed susceptibility patterns similar to that of their parent isolates against the three schizonticidal drugs except Gm/B2 and Gm/H5 Gambian clones were chloroquine-resistant at 0.3427 microM (0.006 mefloquine activity) and 0.2260 microM (0.004 mefloquine activity), respectively. Further results indicated that they were pure clones compared to their parent isolates as their schizonticidal drug susceptibilities were statistically different (p < 0.05) except Gm/C6 and TGR/B7 clones against mefloquine (p < 0.05).
Emergence and spread of resistant parasites to the newest chemotherapeutic anti-malarial agents are the biggest challenges against malaria control programs. Therefore, developing a novel effective treatment to reduce the overgrowing burden of multidrug resistant malaria is a pressing need. Herein, we have developed a biocompatible and biodegradable, non-toxic chitosan-tripolyphosphate-chloroquine (CS-TPP CQ) nanoparticle. CS-TPP CQ nanoparticles effectively kill the parasite through redox generation and induction of the pro- and anti-inflammatory cytokines in both sensitive and resistant parasite in vitro. The in vitro observations showed a strong inhibitory effect (p
Artemether (ARTM) is a very effective antimalarial drug with poor solubility and consequently low bioavailability. Smart nanocrystals of ARTM with particle size of 161±1.5 nm and polydispersity index of 0.172±0.01 were produced in <1 hour using a wet milling technology, Dena(®) DM-100. The crystallinity of the processed ARTM was confirmed using differential scanning calorimetry and powder X-ray diffraction. The saturation solubility of the ARTM nanocrystals was substantially increased to 900 µg/mL compared to the raw ARTM in water (145.0±2.3 µg/mL) and stabilizer solution (300.0±2.0 µg/mL). The physical stability studies conducted for 90 days demonstrated that nanocrystals stored at 2°C-8°C and 25°C were very stable compared to the samples stored at 40°C. The nanocrystals were also shown to be stable when processed at acidic pH (2.0). The solubility and dissolution rate of ARTM nanocrystals were significantly increased (P<0.05) compared to those of its bulk powder form. The results of in vitro studies showed significant antimalarial effect (P<0.05) against Plasmodium falciparum and Plasmodium vivax. The IC50 (median lethal oral dose) value of ARTM nanocrystals was 28- and 54-fold lower than the IC50 value of unprocessed drug and 13- and 21-fold lower than the IC50 value of the marketed tablets, respectively. In addition, ARTM nanocrystals at the same dose (2 mg/kg) showed significantly (P<0.05) higher reduction in percent parasitemia (89%) against P. vivax compared to the unprocessed (27%), marketed tablets (45%), and microsuspension (60%). The acute toxicity study demonstrated that the LD50 value of ARTM nanocrystals is between 1,500 mg/kg and 2,000 mg/kg when given orally. This study demonstrated that the wet milling technology (Dena(®) DM-100) can produce smart nanocrystals of ARTM with enhanced antimalarial activities.
Endophytic Streptomyces strains are potential sources for novel bioactive molecules. In this study, the diketopiperazine gancidin W (GW) was isolated from the endophytic actinobacterial genus Streptomyces, SUK10, obtained from the bark of Shorea ovalis tree, and it was tested in vivo against Plasmodium berghei PZZ1/100. GW exhibited an inhibition rate of nearly 80% at 6.25 and 3.125 μg kg-1 body weight on day four using the 4-day suppression test method on male ICR strain mice. Comparing GW at both concentrations with quinine hydrochloride and normal saline as positive and negative controls, respectively, 50% of the mice treated with 3.125 μg kg-1 body weight managed to survive for more than 11 months after infection, which almost reached the life span of normal mice. Biochemical tests of selected enzymes and proteins in blood samples of mice treated with GW were also within normal levels; in addition, no abnormalities or injuries were found on internal vital organs. These findings indicated that this isolated bioactive compound from Streptomyces SUK10 exhibits very low toxicity and is a good candidate for potential use as an antimalarial agent in an animal model.
Malaria has been teasing human populations from a long time. Presently, several classes of antimalarial drugs are available in market, but the issues of toxicity, lower efficacy and the resistance by malarial parasites have decreased their overall therapeutic indices. Thus, the search for new promising antimalarials continues, however, the battle against malaria is far from over. Ferroquine is a derivative of chloroquine with antimalarial properties. It is the most successful of the chloroquine derivatives. Not only ferroquine, but also its derivatives have shown promising potential as antimalarials of clinical interest. Presently, much research is dedicated to the development of ferroquine derivatives as safe alternatives to antimalarial chemotherapy. The present article describes the structural, chemical and biological features of ferroquine. Several classes of ferroquine derivatives including hydroxyferroquines, trioxaferroquines, chloroquine-bridged ferrocenophanes, thiosemicarbazone derivatives, ferrocene dual conjugates, 4-N-substituted derivatives, and others have been discussed. Besides, the mechanism of action of ferroquine has been discussed. A careful observation has been made into pharmacologically significant ferroquine derivatives with better or equal therapeutic effects to that of chloroquine and ferroquine. A brief discussion of the toxicities of ferroquine derivatives has been made. Finally, efforts have been made to discuss the current challenges and future perspectives of ferroquine-based antimalarial drug development.
Discovery and development of antimalarial drugs have long been dominated by single-target therapy. Continuous effort has been made to explore and identify different targets in malaria parasite crucial for the malaria treatment. The single-target drug therapy was initially successful, but it was later supplanted by combination therapy with multiple drugs to overcome drug resistance. Emergence of resistant strains even against the combination therapy has warranted a review of current antimalarial pharmacotherapy. This has led to the development of the new concept of covalent biotherapy, in which two or more pharmacophores are chemically bound to produce hybrid antimalarial drugs with multi-target functionalities. Herein, the review initially details the current pharmacotherapy for malaria as well as the conventional and novel targets of importance identified in the malaria parasite. Then, the rationale of multi-targeted therapy for malaria, approaches taken to develop the multi-target antimalarial hybrids, and the examples of hybrid molecules are comprehensively enumerated and discussed.
Chloroquine (CQ) and mefloquine (MQ) are no longer potent antimalarial drugs due to the emergence of resistant Plasmodium falciparum. Combination therapy has become the standard for many regimes in overcoming drug resistance. Roxithromycin (ROM), a known p-glycoprotein inhibitor, is reported to have antimalarial activity and it is hoped it will potentiate the effects of both CQ/MQ and reverse CQ/MQ-resistance. We assayed the effects of CQ and MQ individually and in combination with ROM on synchronized P. falciparum (Dd2 strain) cultures. The IC(50) values of CQ and MQ were 60.0+/-5.0 and 16.0+/-3.0 ng/ml; these were decreased substantially when combined with ROM. Isobolograms indicate that CQ-ROM combinations were relatively more synergistic (mean FICI 0.70) than MQ-ROM (mean FICI 0.85) with their synergistic effect at par with CQ-verapamil (VRP) (mean FICI 0.64) and MQ-VRP (mean FICI 0.60) combinations. We conclude that ROM potentiates the CQ/MQ response on multidrug-resistant P. falciparum.
Five Malaysian isolates of the protozoan Plasmodium falciparum Welch were cultured in vitro following the method of Trager and Jensen (1976, 1977) and subsequently cloned using the limiting dilution method of Rosario (1981). Thirty clones were obtained and were later characterized against schizontocidal drugs, chloroquine, mefloquine and quinine, using the modified in vitro microtechnique. Results showed that these local isolates were heterogeneous and most of the clones exhibited similar pattern of susceptibility as their parent isolate except for ST 168 clone and two ST 195 clones that were sensitive but two ST 165 clones, two ST 168 clones and five ST 195 clones were resistant against quinine, respectively. Results also indicated that they were pure clones compared to their parent isolate because their drug susceptibility studies were significantly different (p < 0.05).
Malaria, one of the most deadly diseases of our time affects more than 200 million people across the globe and is responsible for about one million deaths annually. Until recently Plasmodium falciparum has been the main cause for malarial infection in human beings but now Plasmodium knowlesi from Malaysia remains as one of the most virulent parasite spreading fast not only in Malaysia but in different parts of the world. Hence there is urgent need for the global fight to control malaria. Global malaria eradication program by use of insecticide spraying has resulted in good response in the past. Treatment of malaria infected patients with anti-malarial drugs has helped to eliminate malarial infections successfully but with increased resistance displayed by malarial parasites to these drugs there is resurgence of malaria caused both by drug resistance as well as by infection caused by new malarial species like Plasmodium knowlesi. With recent advances on molecular studies on malarial parasites it is now clear that the pineal hormone melatonin acts as a cue for growth and development of Plasmodium falciparum. Same may be true for Plasmodium knowlesi also. Hence treatment modalities that can effectively block the action of melatonin on Plasmodium species during night time by way of using either bright light therapy or use of melatonin receptor blocking can be considered as useful approaches for eliminating malarial infection in man.