Antimalarial proteasome inhibitor reveals collateral sensitivity from intersubunit interactions and fitness cost of resistance.

We describe noncovalent, reversible asparagine ethylenediamine (AsnEDA) inhibitors of the Plasmodium falciparum proteasome (Pf20S) ß5 subunit that spare all active subunits of human constitutive and immuno-proteasomes. The compounds are active against erythrocytic, sexual, and liver-stage parasites, against parasites resistant to current antimalarials, and against P. falciparum strains from patients in Africa. The ß5 inhibitors synergize with a ß2 inhibitor in vitro and in mice and with artemisinin. P. falciparum selected for resistance to an AsnEDA ß5 inhibitor surprisingly harbored a point mutation in the noncatalytic ß6 subunit. The ß6 mutant was resistant to the species-selective Pf20S ß5 inhibitor but remained sensitive to the species-nonselective ß5 inhibitors bortezomib and carfilzomib. Moreover, resistance to the Pf20S ß5 inhibitor was accompanied by increased sensitivity to a Pf20S ß2 inhibitor. Finally, the ß5 inhibitor-resistant mutant had a fitness cost that was exacerbated by irradiation. Thus, used in combination, multistage-active inhibitors of the Pf20S ß5 and ß2 subunits afford synergistic antimalarial activity with a potential to delay the emergence of resistance to artemisinins and each other.

Preclinical Efficacy and Safety Assessment of Artemisinin-Chemotherapeutic Agent Conjugates for Ovarian Cancer.

Artemisinin (ARS) and its derivatives, which are clinically used antimalarial agents, have shown antitumor activities. Their therapeutic potencies, however, are limited by their low solubility and poor bioavailability. Here, through a pharmacophore hybridization strategy, we synthesized ARS-drug conjugates, in which the marketed chemotherapeutic agents chlorambucil, melphalan, flutamide, aminoglutethimide, and doxifluridine, were separately bonded to Dihydroartemisinin (DHA) through various linkages. Of these, the artemisinin-melphalan conjugate, ARS4, exhibited most toxicity to human ovarian cancer cells but had low cytotoxicity to normal cells. ARS4 inhibited the growth and proliferation of ovarian cancer cells and resulted in S-phase arrest, apoptosis, and inhibition of migration; these effects were stronger than those of its parent drugs, DHA and melphalan. Furthermore, ARS4 modulated the expression of proteins involved in cell cycle progression, apoptosis, and the epithelial-mesenchymal transition (EMT). Moreover, in mice, ARS4 inhibited growth and intraperitoneal dissemination and metastasis of ovarian cancer cells without observable toxic effects. Our results provide a basis for development of the compound as a chemotherapeutic agent. RESEARCH IN CONTEXT: Artemisinin compounds have recently received attention as anticancer agents because of their clinical safety profiles and broad efficacy. However, their therapeutic potencies are limited by low solubility and poor bioavailability. Here, we report that ARS4, an artemisinin-melphalan conjugate, possesses marked in-vitro and in-vivo antitumor activity against ovarian cancer, the effects of which are stronger than those for its parent drugs, Dihydroartemisinin and melphalan. In mice, ARS4 inhibits localized growth of ovarian cancer cells and intraperitoneal dissemination and metastasis without appreciable host toxicity. Thus, for patients with ovarian cancer, ARS4 is a promising chemotherapeutic agent.

New orally active diphenylmethyl-based ester analogues of dihydroartemisinin: Synthesis and antimalarial assessment against multidrug-resistant Plasmodium yoelii nigeriensis in mice.

A new series of ester analogues of artemisinin 8a-f, incorporating diphenylmethyl as pharmacologically privileged substructure, and 8g-j have been prepared and evaluated for their antimalarial activity against multidrug-resistant (MDR) Plasmodium yoelii nigeriensis in Swiss mice via oral route. These diphenylmethyl-based ester analogues 8a-f were found to be 2-4 folds more active than the antimalarial drugs ß-arteether 4 and artesunic acid 5. Ester 8a, the most active compound of the series, provided complete protection to the infected mice at 24 mg/kg × 4 days as well as 12 mg/kg × 4 days, respectively. In this model ß-arteether provided 100% and 20% protection at 48 mg/kg × 4 days and 24 mg/kg × 4 days, respectively.

Integration of novel low-cost colorimetric, laser photometric, and visual fluorescent techniques for rapid identification of falsified medicines in resource-poor areas: application to artemether-lumefantrine.

The availability of falsified antimalarial drugs can be reduced with effective drug regulatory agencies and proper enforcement. Fundamental to these agencies taking action, rapid identification must be made as soon as they appear in the market place. Since falsified antimalarials occur mostly in developing countries, performing drug analysis presents itself with unique challenges. A fundamental factor in choosing a useful technique is affordability and simplicity. Therefore, we suggest a three-tiered drug evaluation strategy for identifying a falsified drug in resource-poor areas. Tier I is a simple comparison of a tablet's weight and dimensions with official specifications. Tier II uses inexpensive photometric devices (laser and fluorescence) to evaluate a tablet. Suspicious samples from Tier I and II assessments are then subjected to a colorimetric assay for active ingredients identification and quantification. In this article, we evaluate a novel colorimetric assay for the simultaneous assessment of both lumefantrine and artemether in co-formulated Coartem™ tablets, and integrate the method with two novel, low-cost, fluorescence and laser photometric devices. Image analysis software is used for the assessments. Although artemether-lumefantrine is used as an example, the strategy may be adapted to other medicines.

Design, economical synthesis and antiplasmodial evaluation of vanillin derived allylated chalcones and their marked synergism with artemisinin against chloroquine resistant strains of Plasmodium falciparum.

The in vitro blood stage antiplasmodial activity of a series of allylated chalcones based on the licochalcone A as lead molecule was investigated against chloroquine (CQ) sensitive Pf3D7 and CQ resistant PfINDO strains of Plasmodium falciparum using SYBR Green I assay. Of the forty two chalcones tested, eight showed IC50 ≤ 5 µM. Structure-activity relationship (SAR) studies revealed 9 {1-(4-Chlorophenyl)-3-[3-methoxy-4-(prop-2-en-1-yloxy)phenyl]-prop-2-en-1-one} as the most potent (IC50: 2.5 µM) against Pf3D7 with resistance indices of 1.2 and 6.6 against PfDd2 and PfINDO strains, respectively. Later on, the synergistic effects 9 with standard antimalarials {artemisinin (ART) and chloroquine (CQ)} were studied in order to provide the basis for the selection of the best partner drug. In vitro combinations of 9 with ART showed strong synergy against PfINDO (ΣFIC50: 0.31-0.72) but additive to slight antagonistic effects (ΣFIC50: 1.97-2.64) against Pf3D7. ΣFIC50 0.31 of ART+9 combination corresponded to a 320 fold and 3 fold reduction in IC50 of 9 and ART, respectively. Similar combinations of 9 with CQ showed synergy to additivity to mild antagonism against the two strains {ΣFIC50: 0.668-2.269 (PfINDO); 1.45-2.83 (Pf3D7)}. Drug exposure followed by drug withdrawal indicated that 9 taken alone at IC100 killed rings, trophozoites and schizonts of P. falciparum. The combination of ART and 9 (1X ΣFIC100) selectively inhibited the growth of rings while the 2X ΣFIC100 combination of the same caused killing of rings without affecting trophozoites and schizonts. In contrast, the 1X combination of CQ and 9 (ΣFIC100: 0.5) killed rings and trophozoites. DNA fragmentation and loss of mitochondrial membrane potential (ΔΨm) in the 9 treated P. falciparum culture indicated apoptotic death in malaria parasites. Prediction of ADME properties revealed that most of the molecules did not violate Lipinski's parameters and have low TPSA value suggesting good absorption. The results suggest the promising drug-like properties of 9 against CQ resistant Pf and propensity for synergy with classical antimalarial drugs together with easy and economical synthesis.

High-level semi-synthetic production of the potent antimalarial artemisinin.

In 2010 there were more than 200 million cases of malaria, and at least 655,000 deaths. The World Health Organization has recommended artemisinin-based combination therapies (ACTs) for the treatment of uncomplicated malaria caused by the parasite Plasmodium falciparum. Artemisinin is a sesquiterpene endoperoxide with potent antimalarial properties, produced by the plant Artemisia annua. However, the supply of plant-derived artemisinin is unstable, resulting in shortages and price fluctuations, complicating production planning by ACT manufacturers. A stable source of affordable artemisinin is required. Here we use synthetic biology to develop strains of Saccharomyces cerevisiae (baker's yeast) for high-yielding biological production of artemisinic acid, a precursor of artemisinin. Previous attempts to produce commercially relevant concentrations of artemisinic acid were unsuccessful, allowing production of only 1.6 grams per litre of artemisinic acid. Here we demonstrate the complete biosynthetic pathway, including the discovery of a plant dehydrogenase and a second cytochrome that provide an efficient biosynthetic route to artemisinic acid, with fermentation titres of 25 grams per litre of artemisinic acid. Furthermore, we have developed a practical, efficient and scalable chemical process for the conversion of artemisinic acid to artemisinin using a chemical source of singlet oxygen, thus avoiding the need for specialized photochemical equipment. The strains and processes described here form the basis of a viable industrial process for the production of semi-synthetic artemisinin to stabilize the supply of artemisinin for derivatization into active pharmaceutical ingredients (for example, artesunate) for incorporation into ACTs. Because all intellectual property rights have been provided free of charge, this technology has the potential to increase provision of first-line antimalarial treatments to the developing world at a reduced average annual price.

Substandard artemisinin-based antimalarial medicines in licensed retail pharmaceutical outlets in Ghana.

BACKGROUND & OBJECTIVES: The artemisinin-based antimalarial medicines are first line medicines in the treatment of severe and uncomplicated falciparum malaria. Numerous brands of these medicines manufactured in various countries are available in the Ghanaian market. The study was aimed at evaluating the authenticity and quality of selected brands of artemisinin-based antimalarial medicines marketed in Ghana. METHODS: In all, 14 artemisinin-based antimalarial medicines were purchased from pharmacies (P) and licensed chemical shops (LCSs) in the Kumasi metropolis, Ghana. Simple field tests based on colorimetry and thin layer chromatography were employed in determining the authenticity of the samples. Important quality assessment tests, namely uniformity of mass, crushing strength, disintegration time, and the percentage content of active pharmaceutical ingredients (APIs) were determined. RESULTS: All the brands tested contained the stipulated APIs. Artesunate tablet AT2 failed the uniformity of mass test while artesunate tablets AT3 & AT4 as well as amodiaquine tablets AM4 & AM6 failed the crushing strength test. All the six artemether-lumefantrine tablet brands passed the uniformity of mass, crushing strength and disintegration tests. Only artemether-lumefantrine tablet brand AL1 contained the correct amount of the drugs. The other 13 artemisinin products contained either a lower (underdose) or higher (overdose) amount of the specified drug. Artesunate monotherapy tablets were readily available in pharmacies and licensed chemical shops. INTERPRETATION & CONCLUSION: All the artemisinin-based medicines tested (except AL1) were of substandard quality. The results demonstrate the need for continuous monitoring and evaluation of the quality of artemisininbased antimalarials in the Ghanaian market. Also, the practice of artemisinin antimalarial monotherapy is prevalent in Ghana. Determined efforts should, therefore, be made to eradicate the practice to prevent the development of resistance to the artemisinins.

Self-assembled biotransesterified cyclodextrins as Artemisinin nanocarriers - I: formulation, lyoavailability and in vitro antimalarial activity assessment.

We recently reported a one-step transesterification of cyclodextrins (CDs) by vinyl-acyl fatty esters catalyzed by thermolysin. By using the solvent displacement method and depending on the experimental conditions, the CD derivatives grafted with decanoic alkyl chains (CD-C(10)) yielded either nanosphere or nanoreservoir-type systems with a size ranging from 70 to 220 nm. Both types of nanostructures were able to associate artemisinin (ART), a well-known antimalarial lipophilic drug. The formulation parameters were optimized to reach stable and high ART dosage corresponding to drug levels of 0.3 and 1.6 mg mL(-1) in the colloidal suspension, for the spherical and reservoir-type nanosystems, respectively. PEG surface-decorated nanoparticles were also prepared by co-nanoprecipitation of PEG fatty acid esters and CD-C(10) molecules. The integration of the PEGylated amphiphiles within the CD-C(10) nanostructures did not influence the ART lyoavailability. Both types of ART-loaded nanosystems showed a sustained in vitro release profile over 96 (nanoreservoirs) and 240 h (nanospheres). Finally, the in vitro antimalarial activity was evaluated using the lactate dehydrogenase assay. ART-containing colloidal suspensions inhibited the growth of cultured Plasmodium falciparum, both multi-resistant K1 and susceptible 3D7 strains with IC(50) values (2.8 and 7.0 ng mL(-1)) close to those of reference ART solution. These colloidal nanosystems based on CD derivatives and containing ART may provide a promising alternative formulation for injectable use of ART.

Linker-based hemisuccinate derivatives of artemisinin: synthesis and antimalarial assessment against multidrug-resistant Plasmodium yoelii nigeriensis in mice.

Artesunic acid 5, the hemisuccinate derivative of dihydroartemisinin 2, is the only clinically useful water-soluble derivative of artemisinin 1. However, being a lactol ester, it is rapidly hydrolyzed back to dihydroartemisinin in aqueous alkaline solution, a reaction that seriously limits its utility. A new series of potentially more stable linker-based hemisuccinate derivatives 12a-i and 14a-c have been prepared. The process involved acid-catalyzed reaction of dihydroartemisinin with various diols and polyethylene glycols to give hydroxy-functionalized ethers 7a-i and 10a-c and their further derivatization to hemisuccinate esters 12a-i and 14a-c. Both the hydroxy-functionalized ethers 7a-i and 10a-c and their hemisuccinate derivatives 12a-i and 14a-c have been assessed for antimalarial activity against multidrug-resistant Plasmodium yoelii nigeriensis in Swiss mice. Several of these hemisuccinate derivatives have shown very promising activity. Hemisuccinate derivatives 12f and 12i, the two most active compounds of the series, provided 100% protection to malaria-infected mice at 24 mg/kg × 4 days and therefore are twice as potent as artesunic acid, which provides a similar level of protection at 48 mg/kg × 4 days.

Application of the linear interaction energy method for rational design of artemisinin analogues as haeme polymerisation inhibitors.

The anti-malarial activity of artemisinin-derived drugs appears to be mediated by an interaction of the drug's endoperoxide bridge with intra-parasitic haeme. The binding affinity of artemisinin analogues with haeme were computed using linear interaction energy with a surface generalised Born (LIE-SGB) continuum solvation model. Low levels of root mean square error (0.348 and 0.415 kcal/mol) as well as significant correlation coefficients (r(2) = 0.868 and 0.892) between the experimental and predicted free energy of binding (FEB) based on molecular dynamics and hybrid Monte Carlo sampling techniques establish the SGB-LIE method as an efficient tool for generating more potent inhibitors of haeme polymerisation inhibition.

First assessment in humans of the safety, tolerability, pharmacokinetics, and ex vivo pharmacodynamic antimalarial activity of the new artemisinin derivative artemisone.

In preclinical studies, artemisone (BAY 44-9585), a new artemisinin derivative, was shown to possess enhanced efficacy over artesunate, and it does not possess the neurotoxicity characteristic of the current artemisinins. In a phase I program with double-blind, randomized, placebo-controlled, single and multiple ascending oral-dose studies, we evaluated the safety, tolerability, pharmacokinetics, and ex vivo pharmacodynamic antimalarial activity of artemisone. Single doses (10, 20, 30, 40, and 80 mg) and multiple doses (40 and 80 mg daily for 3 days) of artemisone were administered orally to healthy subjects. Plasma concentrations of artemisone and its metabolites were measured by liquid chromatography/tandem mass spectrometry (LC/MS-MS). Artemisone was well tolerated, with no serious adverse events and no clinically relevant changes in laboratory and vital parameters. The pharmacokinetics of artemisone over the 10- to 80-mg range demonstrated dose linearity. After the single 80-mg dose, artemisone had a geometric mean maximum concentration of 140.2 ng/ml (range, 86.6 to 391.0), a short elimination half-life (t(1/2)) of 2.79 h (range, 1.56 to 4.88), a high oral clearance of 284.1 liters/h (range, 106.7 to 546.7), and a large volume of distribution of 14.50 liters/kg (range, 3.21 to 51.58). Due to artemisone's short t(1/2), its pharmacokinetics were comparable after single and multiple dosing. Plasma samples taken after multiple dosing showed marked ex vivo pharmacodynamic antimalarial activities against two multidrug-resistant Plasmodium falciparum lines. Artemisone equivalent concentrations measured by bioassay revealed higher activity than artemisone measured by LC/MS-MS, confirming the presence of active metabolites. Comparable to those of other artemisinin's, artemisone's t(1/2) is well suited for artemisinin-based combination therapy for the treatment of P. falciparum malaria.

Microbially derived artemisinin: a biotechnology solution to the global problem of access to affordable antimalarial drugs.

Despite considerable efforts by multiple governmental and nongovernmental organizations to increase access to artemisinin-based combination therapies (ACTs), these life-saving antimalarial drugs remain largely unaffordable to the most vulnerable populations. The cost of artemisinin derivatives, ACTs' crucial active ingredients, contributes significantly to the high price of these therapies. With a grant from the Bill and Melinda Gates Foundation, a partnership between Amyris Biotechnologies, the Institute for OneWorld Health, and the University of California, Berkeley is using synthetic biology to help reduce the cost of artemisinin. This article presents a description of the technological platform the partnership--called the Artemisinin Project--is developing to manufacture a low-cost, semi-synthetic artemisinin through a fermentation process. By making life-saving ACTs affordable to the people who most need them, the Artemisinin Project hopes to show that the power of biotechnology can be harnessed to provide solutions to global health problems.

Comparative assessment of technologies for extraction of artemisinin.

This paper describes results of a multiobjective comparative assessment of several established and emerging technologies for extraction of a natural antimalarial substance, artemisinin. Extractions by hexane, supercritical carbon dioxide, hydrofluorocarbon HFC-134a, ionic liquids, and ethanol were considered. Hexane extraction is an established technology and appears to be the most cost-effective. However, it is characterized by lower rates and efficiency of extraction than all other considered techniques and is also worse in terms of safety and environmental impact. Similarly, EtOH extraction was found to be worse than hexane in all assessment parameters. The new technologies (scCO2, HFC, and ILs) are based on nonflammable solvents and are characterized by faster extraction cycles and more complete extraction of the useful substances and enable continuous extraction processes with reduced solvent inventory. Ionic liquid and HFC-134a technologies show considerable promise and should be able to compete with hexane extraction in terms of cost-effectiveness following due process optimization. New technologies are also considerably safer (no risk of explosions, low toxicity) and greener (having a lower environmental impact in use, potential for biodegradability after use). The methodology of comparative assessment of established and emerging technologies is discussed.

Production of the antimalarial drug precursor artemisinic acid in engineered yeast.

Malaria is a global health problem that threatens 300-500 million people and kills more than one million people annually. Disease control is hampered by the occurrence of multi-drug-resistant strains of the malaria parasite Plasmodium falciparum. Synthetic antimalarial drugs and malarial vaccines are currently being developed, but their efficacy against malaria awaits rigorous clinical testing. Artemisinin, a sesquiterpene lactone endoperoxide extracted from Artemisia annua L (family Asteraceae; commonly known as sweet wormwood), is highly effective against multi-drug-resistant Plasmodium spp., but is in short supply and unaffordable to most malaria sufferers. Although total synthesis of artemisinin is difficult and costly, the semi-synthesis of artemisinin or any derivative from microbially sourced artemisinic acid, its immediate precursor, could be a cost-effective, environmentally friendly, high-quality and reliable source of artemisinin. Here we report the engineering of Saccharomyces cerevisiae to produce high titres (up to 100 mg l(-1)) of artemisinic acid using an engineered mevalonate pathway, amorphadiene synthase, and a novel cytochrome P450 monooxygenase (CYP71AV1) from A. annua that performs a three-step oxidation of amorpha-4,11-diene to artemisinic acid. The synthesized artemisinic acid is transported out and retained on the outside of the engineered yeast, meaning that a simple and inexpensive purification process can be used to obtain the desired product. Although the engineered yeast is already capable of producing artemisinic acid at a significantly higher specific productivity than A. annua, yield optimization and industrial scale-up will be required to raise artemisinic acid production to a level high enough to reduce artemisinin combination therapies to significantly below their current prices.

Orally active 1,2,4-trioxanes: synthesis and antimalarial assessment of a new series of 9-functionalized 3-(1-arylvinyl)-1,2,5-trioxaspiro[5.5]undecanes against multi-drug-resistant plasmodium yoelii nigeriensis in mice.

Using easily accessible keto-trioxanes 7a-g as the starting materials, a series of new variously functionalized 1,2,4-trioxanes 10-36 have been prepared and evaluated for antimalarial activity against multi-drug-resistant Plasmodium yoelii nigeriensis in mice in the dose range of 24 mg/kg x 4 days to 96 mg/kg x 4 days by oral route. Trioxanes 10, 12, 14, 16, 18, 20, and 22 have shown promising antimalarial activity. Trioxanes 14 and 18, the two most active compounds of the series, provide 100% and 60% protection at 48 mg/kg x 4 days and 24 mg/kg x 4 days, respectively. In this model beta-arteether provides 100% and 20% protection at 48 mg/kg x 4 days and 24 mg/kg x 4 days, respectively.

Drug resistance hampers our capacity to roll back malaria.

Widespread drug resistance in parasites aggravates the burden of malaria. The extent of the problem is due mainly to the limited armamentarium of drugs used thus far to treat malaria and to policies and practices constrained by limited resources. All drugs in use are affected except, thus far, artemisinin derivatives. The scale and impact of resistance has been underestimated, leading to the continued use of failing drugs, which contributes to the rise in resistance and increased morbidity and mortality due to malaria. Pharmacological, epidemiological, and operational aspects factor the development and spread of resistance. Although the problem is complex, much can be done to reverse the course of events: adopt adequate tests to assess resistance, encourage and sustain development of new drugs, protect drugs against resistance through use of combinations, expand access to prompt and effective treatment, and promote evidence-based policies and sensible practices. The current situation favors the development of sensible strategies to restrain resistance.