Combined antimalarial therapy using artemisinin

The existing armamentarium of drugs for the treatment and prevention of malaria is limited primarily by resistance (and cross-resistance between closely related drugs). However, most of these drugs still have a place and their life-span could be prolonged if better deployed and used, and also by rationally combining them based on pharmacodynamic and pharmacokinetic properties. Newer compounds are also being developed. The nature of malaria disease and its prevalence in the developing world call for innovative approaches to develop new affordable drugs and to safeguard the available ones. According to WHO, the concept of combination therapy is based on the synergistic or additive potential of two or more drugs, to improve therapeutic efficacy and also delay the development of resistance to the individual components of the combination. Combination therapy (CT) with antimalarial drugs is the simultaneous use of two or more blood schizontocidal drugs with independent modes of action and different biochemical targets in the parasite. In the context of this definition, multiple-drug therapies that include a nonantimalarial drug to enhance the antimalarial effect of a blood schizontocidal drug are not considered combination therapy. Similarly, certain antimalarial drugs that fit the criteria of synergistic fixed-dose combinations are operationally considered as single products in that neither of the individual components would be given alone for anti-malarial therapy. An example is sulfadoxine-pyrimethamine. Artemisinin-based combination therapies have been shown to improve treatment efficacy and also contain drug resistance in South-East Asia. However, major challenges exist in the deployment and use of antimalarial drug combination therapies, particularly in Africa. These include: 1) the choice of drug combinations best suited for the different epidemiological situations; 2) the cost of combination therapy; 3) the timing of the introduction of combination therapy; 4) the operational obstacles to implementation, especially compliance. As a response to increasing levels of antimalarial resistance, the World Health Organization (WHO) recommends that all countries experiencing resistance to conventional monotherapies, such as chloroquine, amodiaquine or sulfadoxine/pyrimethamine, should use combination therapies, preferably those containing artemisinin derivatives (ACTs--artemisinin-based combination therapies) for malaria caused by Plasmodium falciparum. There is a promising role of such compounds in replacing or complementing current options. Since 1979, several different formulations of artemisinin and its derivatives have been produced and studied in China in several thousand patients for either P. falciparum or P. vivax malaria. To date, there is no evidence of drug resistance to these compounds. The use of artemisinin, artemether, arteether and artesunate for either uncomplicated or severe malaria is now spreading through almost all malarious areas of the world, although some of they have no patent protection, their development (with few exceptions) has not followed yet full international standards. Both artesunate, artemether and arteether are rapidly and extensively converted to their common bioactive metabolite, dihydroarte-misinin. WHO currently recommends the following therapeutic options: 1) artemether/lumefantrine; 2) artesunate plus amodiaquine; 3) artesunate plus sulfadoxine/pyrimethamine (in areas where SP efficacy remains high); 4) artesunate plus mefloquine (in areas with low to moderate transmission); and 5) amodiaquine plus sulfadoxine/pyrimethamine, in areas where efficacy of both amodiaquine and sulfadoxine/pyrimethamine remains high (mainly limited to countries in West Africa). This non artemisinin-based combination therapy is reserved as an interim option for countries, which, for whatever reason, are unable immediately to move to ACTs.     

Delaying antimalarial drug resistance with combination chemotherapy

Resistance to antimalarial drugs arises when spontaneously occurring mutants with gene mutations or amplifications which confer reduced drug susceptibility are selected, and are then transmitted. Simultaneous use of two or more antimalarials with different modes of action and which therefore do not share the same resistance mechanisms will reduce the chance of selection, because the chance of a resistant mutant surviving is the product of the parasite mutation rates for the individual drugs, multiplied by the number of parasites in an infection that are exposed to the drugs. The artemisinin derivatives are very active antimalarials, which produce large reductions in parasite biomass per asexual cycle, and reduce malaria transmissibility. To date no resistance to these drugs has been reported. These drugs therefore make particularly effective combination partners. This suggests that antimalarial drugs should not be used alone in treatment, but always in combination, as in the treatment of tuberculosis or HIV, and that the combination should include artemisinin or one of its derivatives.     

Evidence for the contribution of the hemozoin synthesis pathway of the murine Plasmodium yoelii to the resistance to artemisinin-related drugs

Plasmodium falciparum malaria is a major global health problem, causing approximately 780,000 deaths each year. In response to the spreading of P. falciparum drug resistance, WHO recommended in 2001 to use artemisinin derivatives in combination with a partner drug (called ACT) as first-line treatment for uncomplicated falciparum malaria, and most malaria-endemic countries have since changed their treatment policies accordingly. Currently, ACT are often the last treatments that can effectively and rapidly cure P. falciparum infections permitting to significantly decrease the mortality and the morbidity due to malaria. However, alarming signs of emerging resistance to artemisinin derivatives along the Thai-Cambodian border are of major concern. Through long-term in vivo pressures, we have been able to select a murine malaria model resistant to artemisinins. We demonstrated that the resistance of Plasmodium to artemisinin-based compounds depends on alterations of heme metabolism and on a loss of hemozoin formation linked to the down-expression of the recently identified Heme Detoxification Protein (HDP). These artemisinins resistant strains could be able to detoxify the free heme by an alternative catabolism pathway involving glutathione (GSH)-mediation. Finally, we confirmed that artemisinins act also like quinolines against Plasmodium via hemozoin production inhibition. The work proposed here described the mechanism of action of this class of molecules and the resistance to artemisinins of this model. These results should help both to reinforce the artemisinins activity and avoid emergence and spread of endoperoxides resistance by focusing in adequate drug partners design. Such considerations appear crucial in the current context of early artemisinin resistance in Asia.     

Artemisinin, an endoperoxide antimalarial, disrupts the hemoglobin catabolism and heme detoxification systems in malarial parasite.

Endoperoxide antimalarials based on the ancient Chinese drug Qinghaosu (artemisinin) are currently our major hope in the fight against drug-resistant malaria. Rational drug design based on artemisinin and its analogues is slow as the mechanism of action of these antimalarials is not clear. Here we report that these drugs, at least in part, exert their effect by interfering with the plasmodial hemoglobin catabolic pathway and inhibition of heme polymerization. In an in vitro experiment we observed inhibition of digestive vacuole proteolytic activity of malarial parasite by artemisinin. These observations were further confirmed by ex vivo experiments showing accumulation of hemoglobin in the parasites treated with artemisinin, suggesting inhibition of hemoglobin degradation. We found artemisinin to be a potent inhibitor of heme polymerization activity mediated by Plasmodium yoelii lysates as well as Plasmodium falciparum histidine-rich protein II. Interaction of artemisinin with the purified malarial hemozoin in vitro resulted in the concentration-dependent breakdown of the malaria pigment. Our results presented here may explain the selective and rapid toxicity of these drugs on mature, hemozoin-containing, stages of malarial parasite. Since artemisinin and its analogues appear to have similar molecular targets as chloroquine despite having different structures, they can potentially bypass the quinoline resistance machinery of the malarial parasite, which causes sublethal accumulation of these drugs in resistant strains.     

Factors affecting the pharmacokinetics and pharmacodynamics of liposomal drugs

Various attempts to increase the therapeutic index of the drug while minimizing side effects have been made in drug delivery systems. Among several promising strategies, liposomes represent an advanced technology to target active molecules to the site of action. Rapid clearance of circulating liposomal drugs administered intravenously has been a critical issue because circulation time in the blood affects drug exposure at the target site. The clinical use of liposomal drugs is complicated by large intra- and interindividual variability in their pharmacokinetics (PK) and pharmacodynamics (PD). Thus, it is important to understand the factors affecting the PK/PD of the liposomal formulation of drugs and to elucidate the mechanisms underlying the variability in the PK/PD of liposomal drugs. In this review article, we describe the characteristics of liposome formulations and discuss the effects of various factors, including liposome-associated factors, host-associated factors, and treatment on the PK/PD of liposomal agents.     

Factors affecting the pharmacokinetics and pharmacodynamics of liposomal drugs

Various attempts to increase the therapeutic index of the drug while minimizing side effects have been made in drug delivery systems. Among several promising strategies, liposomes represent an advanced technology to target active molecules to the site of action. Rapid clearance of circulating liposomal drugs administered intravenously has been a critical issue because circulation time in the blood affects drug exposure at the target site. The clinical use of liposomal drugs is complicated by large intra- and interindividual variability in their pharmacokinetics (PK) and pharmacodynamics (PD). Thus, it is important to understand the factors affecting the PK/PD of the liposomal formulation of drugs and to elucidate the mechanisms underlying the variability in the PK/PD of liposomal drugs. In this review article, we describe the characteristics of liposome formulations and discuss the effects of various factors, including liposome-associated factors, host-associated factors, and treatment on the PK/PD of liposomal agents.     

Novel anti-malarial drug strategies to prevent artemisinin partner drug resistance: A model-based analysis

Emergence of resistance to artemisinin and partner drugs in the Greater Mekong Subregion has made elimination of malaria from this region a global priority; it also complicates its achievement. Novel drug strategies such as triple artemisinin combination therapies (ACTs) and chemoprophylaxis have been proposed to help limit resistance and accelerate elimination. The objective of this study was to better understand the potential impacts of triple ACTs and chemoprophylaxis, using a mathematical model parameterized using data from Cambodia. We used a simple compartmental model to predict trends in malaria incidence and resistance in Cambodia from 2020–2025 assuming no changes in transmission since 2018. We assessed three scenarios: a status quo scenario with artesunate-mefloquine (ASMQ) as treatment; a triple ACT scenario with dihydroartemisinin-piperaquine (DP) plus mefloquine (MQ) as treatment; and a chemoprophylaxis scenario with ASMQ as treatment plus DP as chemoprophylaxis. We predicted MQ resistance to increase under the status quo scenario. Triple ACT treatment reversed the spread of MQ resistance, but had no impact on overall malaria incidence. Joint MQ-PPQ resistance declined under the status quo scenario for the baseline parameter set and most sensitivity analyses. Compared to the status quo, triple ACT treatment limited spread of MQ resistance but also slowed declines in PPQ resistance in some sensitivity analyses. The chemoprophylaxis scenario decreased malaria incidence, but increased the spread of strains resistant to both MQ and PPQ; both effects began to reverse after the intervention was removed. We conclude that triple ACTs may limit spread of MQ resistance in the Cambodia, but would have limited impact on malaria incidence and might slow declines in PPQ resistance. Chemoprophylaxis could have greater impact on incidence but also carries higher risks of resistance. Aggressive strategies to limit transmission the GMS are needed to achieve elimination goals, but any intervention should be accompanied by monitoring for drug resistance.     

Novel polymorphisms in Plasmodium falciparum ABC transporter genes are associated with major ACT antimalarial drug resistance

Chemotherapy is a critical component of malaria control. However, the most deadly malaria pathogen, Plasmodium falciparum, has repeatedly mounted resistance against a series of antimalarial drugs used in the last decades. Southeast Asia is an epicenter of emerging antimalarial drug resistance, including recent resistance to the artemisinins, the core component of all recommended antimalarial combination therapies. Alterations in the parasitic membrane proteins Pgh-1, PfCRT and PfMRP1 are believed to be major contributors to resistance through decreasing intracellular drug accumulation. The pfcrt, pfmdr1 and pfmrp1 genes were sequenced from a set of P.falciparum field isolates from the Thai-Myanmar border. In vitro drug susceptibility to artemisinin, dihydroartemisinin, mefloquine and lumefantrine were assessed. Positive correlations were seen between the in vitro susceptibility responses to artemisinin and dihydroartemisinin and the responses to the arylamino-alcohol quinolines lumefantrine and mefloquine. The previously unstudied pfmdr1 F1226Y and pfmrp1 F1390I SNPs were associated significantly with artemisinin, mefloquine and lumefantrine in vitro susceptibility. A variation in pfmdr1 gene copy number was also associated with parasite drug susceptibility of artemisinin, mefloquine and lumefantrine. Our work unveils new candidate markers of P. falciparum multidrug resistance in vitro, while contributing to the understanding of subjacent genetic complexity, essential for future evidence-based drug policy decisions.     

Current perspectives on the mechanism of action of artemisinins

Artemisinin derivatives are the most recent single drugs approved and introduced for public antimalarial treatment. Although their recommended use is for treatment of Plasmodium falciparum infection, these drugs also act against other parasites, as well as against tumor cells. The mechanisms of action attributed to artemisinin include interference with parasite transport proteins, disruption of parasite mitochondrial function, modulation of host immune function and inhibition of angiogenesis. Artemisinin combination therapies are currently the preferred treatment for malaria. These combinations may prevent the induction of parasite drug resistance. However, in view of the multiple mechanisms involved, especially when additional drugs are used, the combined therapy should be carefully examined for antagonistic effects. It is now a general theory that the crucial mechanism is interference with plasmodial SERCA. Therefore, future development of resistance may be associated with overproduction or mutations of this transporter. However, a general mechanism, such as alterations in general drug transport pathways, is feasible. In this article, we review the evidence for each mechanism of action suggested.     

Dried Whole Plant Artemisia annua as an Antimalarial Therapy

Drugs are primary weapons for reducing malaria in human populations. However emergence of resistant parasites has repeatedly curtailed the lifespan of each drug that is developed and deployed. Currently the most effective anti-malarial is artemisinin, which is extracted from the leaves of Artemisia annua. Due to poor pharmacokinetic properties and prudent efforts to curtail resistance to monotherapies, artemisinin is prescribed only in combination with other anti-malarials composing an Artemisinin Combination Therapy (ACT). Low yield in the plant, and the added cost of secondary anti-malarials in the ACT, make artemisinin costly for the developing world. As an alternative, we compared the efficacy of oral delivery of the dried leaves of whole plant (WP) A. annua to a comparable dose of pure artemisinin in a rodent malaria model (Plasmodium chabaudi). We found that a single dose of WP (containing 24 mg/kg artemisinin) reduces parasitemia more effectively than a comparable dose of purified drug. This increased efficacy may result from a documented 40-fold increase in the bioavailability of artemisinin in the blood of mice fed the whole plant, in comparison to those administered synthetic drug. Synergistic benefits may derive from the presence of other anti-malarial compounds in A. annua. If shown to be clinically efficacious, well-tolerated, and compatible with the public health imperative of forestalling evolution of drug resistance, inexpensive, locally grown and processed A. annua might prove to be an effective addition to the global effort to reduce malaria morbidity and mortality.     

A network to monitor antimalarial drug resistance: a plan for moving forward

The spread of resistance to antimalarial drugs has required changes in the recommended first-line treatment for falciparum malaria in almost all regions. Most drugs recommended currently are combinations of a long-acting antimalarial and an artemisinin derivative. This article presents the rationale for establishing a web-based, open-access database of antimalarial drug resistance and efficacy: the World Antimalarial Resistance Network (WARN). The goal of this network is to assemble the tools and information that will enable the malaria community to collate, analyze and share contemporary information on antimalarial-drug efficacy in all endemic regions so that decisions on antimalarial-drug use are based on solid evidence.     

The potentiating action of tetrandrine in combination with chloroquine or qinghaosu against chloroquine-sensitive and resistant falciparum malaria

Using chloroquine-sensitive (CS) and chloroquine-resistant (CR) strains of Plasmodium falciparum in vitro, interactions between tetrandrine (TT) and either chloroquine (CQ) or qinghaosu (QHS, artemisinin) were assessed using isobolograms. Sums of the fractional inhibitory concentration for the combination of the two drugs are less than one and therefore, we can conclude that in vitro TT and CQ or QHS act synergistically against CS and CR falciparum malaria. Remarkably, using CR malaria, TT can lower the IC50 dose of CQ as much as 40 fold. These drug combinations may impair the advantage that the development of CQ resistance conveys on the parasite.     

Antimalarial drug resistance and combination chemotherapy.

Antimarial drug resistance develops when spontaneously occurring parasite mutants with reduced susceptibility are selected, and are then transmitted. Drugs for which a single point mutation confers a marked reduction in susceptibility are particularly vulnerable. Low clearance and a shallow concentration-effect relationship increase the chance of selection. Use of combinations of antimalarials that do not share the same resistance mechanisms will reduce the chance of selection because the chance of a resistant mutant surviving is the product of the per parasite mutation rates for the individual drugs, multiplied by the number of parasites in an infection that are exposed to the drugs. Artemisinin derivatives are particularly effective combination partners because (i) they are very active antimalarials, producing up to 10,000-fold reductions in parasite biomass per asexual cycle; (ii) they reduce malaria transmissibility; and (iii) no resistance to these drugs has been reported yet. There are good arguments for no longer using antimalarial drugs alone in treatment, and instead always using a combination with artemisinin or one of its derivatives.     

Plasmodium sensitivity to artemisinins: magic bullets hit elusive targets

Artemisinins are efficacious antimalarial drugs widely employed as first-line treatment in endemic countries under the form of combined therapies. Different molecular modes of action have been postulated to explain the parasiticidal effect of these compounds; however, none has been unequivocally accepted, and their physiological relevance is still questioned. Similarly, no definite genetic determinant of Plasmodium sensitivity to artemisinins has been identified so far. A better understanding of the mode of action of artemisinins and the genetic basis of laboratory-induced or field-observed altered susceptibility is crucial for malaria control. In this review different models of artemisinins' molecular action are briefly presented, focusing on recent advances, and the evidence of potential association between various gene polymorphisms and artemisinin resistance is comprehensively reviewed.     

Artemisinin: mechanisms of action,resistance and toxicity

Artemisinin and its derivatives are widely used throughout the world. The mechanism of action of these compounds appears to involve the heme-mediated decomposition of the endoperoxide bridge to produce carbon-centred free radicals. The involvement of heme explains why the drugs are selectively toxic to malaria parasites. The resulting carbon-centred free radicals are alkylate heme and proteins, one of which is the translationally controlled tumour protein. Clinically relevant artemisinin resistance has not been demonstrated, but it is likely to occur since artemisinin resistance has been obtained in laboratory models. At high doses, artemisinin can be neurotoxic but toxicity has not been found in clinical studies. The mechanism of neurotoxicity may be similar to the mechanism of action.     

Combination therapy as a strategy to prevent antimalarial drug resistance

Resistance of Plasmodium falciparum to antimalarials is considered one of the factors responsible for the impairment of the malaria treatment and control worldwide. Resistance emerges as a result of selection and then disemination of spontaneous mutant parasites with reduced drug susceptibility. Combination therapy is considered as the main strategy to control antimalarial drug resistance. Currently, combination therapies that include artemisinin derivatives are highly recommended. Combination therapy has been used in Colombia for more than 20 years; however, its impact on preventing the dissemination of drug resistance is unknown. This paper reviews the theoretical bases and clinical studies that support the use of combination therapy.     

Plasmodium falciparum malaria: rosettes are disrupted by quinine, artemisinin, mefloquine, primaquine, pyrimethamine, chloroquine and proguanil.

An assay was developed measuring the disruption of rosettes between Plasmodium falciparuminfected (trophozoites) and uninfected erythrocytes by the antimalarial drugs quinine, artemisinin mefloquine, primaquine, pyrimethamine, chloroquine and proguanil. At 4 hr incubation rosettes were disrupted by all the drugs in a dose dependent manner. Artemisinin and quinine were the most effective anti-malarials at disrupting rosettes at their therapeutic concentrations with South African RSA 14, 15, 17 and The Gambian FCR-3 P. falciparum strains. The least effective drugs were proguanil and chloroquine. A combination of artemisinin and mefloquine was more effective than each drug alone. The combinations of pyrimethamine or primaquine, with quinine disrupted more rosettes than quinine alone. Quinine may be an effective drug in the treatment of severe malaria because the drug efficiently reduces the number of rosettes.     

Antimalarial drug quality in the most severely malarious parts of Africa - a six country study

A range of antimalarial drugs were procured from private pharmacies in urban and peri-urban areas in the major cities of six African countries, situated in the part of that continent and the world that is most highly endemic for malaria. Semi-quantitative thin-layer chromatography (TLC) and dissolution testing were used to measure active pharmaceutical ingredient content against internationally acceptable standards. 35% of all samples tested failed either or both tests, and were substandard. Further, 33% of treatments collected were artemisinin monotherapies, most of which (78%) were manufactured in disobservance of an appeal by the World Health Organisation (WHO) to withdraw these clinically inappropriate medicines from the market. The high persistence of substandard drugs and clinically inappropriate artemisinin monotherapies in the private sector risks patient safety and, through drug resistance, places the future of malaria treatment at risk globally.     

Plasmodium falciparum: in vitro interactions of artemisinin with amodiaquine, pyronaridine, and chloroquine

In the scenario of drug-resistant Plasmodium falciparum malaria combination therapy represents an effective approach. Artemisinin and its derivatives are of special interest because they represent the most effective group of compounds against multidrug-resistant malaria with a rapid onset of action and a short half-life. Interactions of artemisinin with amodiaquine, pyronaridine, and chloroquine were therefore investigated against three strains of P. falciparum using a 48-h in vitro culture assay. Two of the strains were chloroquine sensitive and one was partially chloroquine resistant. Observed effective concentrations (O) of the combined compounds at different concentration ratios were calculated for different degrees of inhibition (EC50, EC90, EC99) and compared to expected calculated effective concentrations (E) using a probit method. Synergism with mean O/E EC90 values of 0.25 and 0.8 were found with the combination of artemisinin and the two Mannich bases, amodiaquine and pyronaridine, respectively, whereas chloroquine showed addition with a mean value of 1.2. Although both amodiaquine and chloroquine are 4-aminoquinolines, their interaction with artemisinin appears to be different. The combination of artemisinin with amodiaquine represents an important option for the treatment of falciparum malaria.