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The digestive vacuole and malaria resistance ; the weakness of artesunate.

January 9, 2014 - 11:04 -- Pierre Lutgen

The diffusion of antimalarials into infected red blood cells has been studied by several authors. For example, three times more chloroquine accumulates in CQ sensitive strains than in the CQ resistant ones (KJ Saliba et al., Biochem Pharmacol. 1998, Aug 1 ;56, 313-20) Thediffusion of artemisinin into parasitized RBCs was found to be rapid, saturable, temperature dependent, irreversible. In contrast, simple passive diffusion of artemisinin was seen in nonparasitized RBCs.(N Vyas, Antimicro Ag Chemother, Jan 2002, 105-109). But it was also 43% lower in resistant strains than in sensitive strains of Plasmodium yoelii (D Walkeret al., Antimicrob Ag Chemotherapy Feb 2000, 344-347). The partitioning is subject to competitive inhibition. The cross resistance between artemisinin and other antimalarials has been described already 20 years ago (Doury JC, Ringwald P et al., Trop Med Parasitol 1992, 43 :3 197-8). Comparing monotherapy with the combined action of artesunate and amodiaquine it was found that the total exposure to both drugs was reduced significantly when they were given in combination, 67 % lower for artesunate and 65% for amodiaquine (C Orrell et al., Eur J Clin Pharmacol , 64. 683,90, 2008). Other molecules present in the Artemisia plant also may interfer with the artemisinin uptake into the RBC as they do for glucose needed by the parasite for the anaerobic glycolysis.Chalcones inhibit permeation pathways induced by the parasite in the host erythrocyte membrane (ML Go Antimicro Ag Chemotherapy Sept 2004, 3241-45). Some Artemisia annua genotypes like the one from Luxembourg are rich in artemisia ketones.

Inside the RBC artemisinin or other antimalarials are transported to the DV by hemoglobin transport vehicles. The accumulation of the antimalarial in the DV depends on many factors. Hemozoin nucleation occurs at the DV inner membrane. The effect of antimalarials after penetration into the parasite and the DV may vary. Some like artesunate abolish the movement of the trophozoite and the malaria pigment, others like quinine and piperaquine do not. (J Wongtanachei et al., Southeast Asian J Trop Med Public Health. 2012 Jan;43:1, 1-9). They may finally lead to a destruction of the DV membrane and total desorganisation of the parasites.

The human malaria parasite has a homologue of the human multidrug resistance P-glycoprotein that pumps drugs from the cancer cells. The MDR1-Pgh1protein is localized on the digestive vacuole (A Cowman et al., J Cell Biology, 113 :5 1999, 1033-1042).Pgh1 influences the sensitivity of malaria parasites to a diverse range of antimalarial drugs, reducing their concentration at the site of action. But the efflux can be inhibited by other drugs, verapamil for example. Chloroquine accumulation in erythrocytes is energy dependant. After glucose addition the uptake is markedly different in CQR and CQS strains. It decreases in the first and increases in the second (CP Sanchez et al., Biochemistry 42, 9283-94). Mutations in the Pgh1 can confer resistance to mefloquine, quinine and eventually to the structurally unrelated compound artemisinin. In the jejunum artemisinin is not an inducer of P-glycoprotein and the permeability of artemisinin remains high after multiple, low or high, doses. (U Svensson et al., Drug Metabolism and Disposition 27.2, 1999, 227-232).

Like bioavailability or permeability, instability of artemisinin in water or plasma has evolved from a rumour to a dogma. Several papers show that artemisinin can be extracted up to 90 % in tea infusions and stays stable for at least 24 hours (Fr van der Kooy et al., Planta Med. 2011 Oct;77(15):1754-6), (AC Beekman et al., J Pharm Pharmacol 1997, 49 :12, 1254-8).

A German team has compared the pathways of 21 selected antimalarials and their metabolites. (A Burk et al., Brit J Pharmacology, 2012, 167, 666-671). It investigated their effect on 2 xenosensing receptors CAR and PXR, but also on CYP3A4, Caco-2 cells and MDR1/Pgh1 efflux pump. Their results are surprising : Only artemisinin is converted to an enzyme-inducing metabolite, the ostracized deoxyartemisinin. Artesunate was the only drug of the artemisin class scoring lower for its agonist properties. Even at 300 microM artesunate and DHA did not show a coactivator interaction. All antimalarials, except artemisinin and its derivatives, were cytotoxic at 100 microM. The properties of metabolites like deoxyartemisinin (persistance of induction for five days) might explain the action of artemisinin in vivo.

An Italian team recently found that dihydroartemisinin (DHA), a metabolite of artesunate, induces significant depletion of early embryonic erythroblasts in animal models. Artemisinin does not generate DHA but only the metabolite deoxyartemisinin which did not have this erythro-toxic effect which could be harmful for stem cells during pregnancy ( S Finaunini et al., Toxicology, 2012, 300, 57-66)

The DV is an important site of antimalarial activity. The destruction of the food vacuole membrane leads to a total desorganization of the parasites (Y Maeno et al.,Am J Trop Med Hyg, 1993, 49 :4, 485-91). It leaves individual hemozoin crystals in direct contact with the parasite cytoplasm. The action of artemisinin on disruption of DV membrane has been compared to that of novel synthetic endoperoxides (Crespo et al., Antimicrob Agents and Chemother., 52.1, 2008, 98-109). The latter do not. This is understandable, the pharmacokinetics and pharmacodynamics of artesunate and artemisinin are completely different. The first desintegrates into the very reactive DHA, the second into molecules which have lost their peroxide bridge and show now antiplasmodial activity, at least in vitro. But the two have been compared very little in vivo. No clinical trial on humans has been run with pure artemisinin over the last 30 years.

Recently (D.Wilson et al., Antimicrob Ag Chemotherapy, 2013, 57-3, 1455-67) studied the timing of action of quinine and artemisinin derivatives against Plasmodium falciparum. Artemisinin is the only one which prevents the rupture of schizonts and the realease of merozoites. Artesunate and chloroquine and all others do not. As the authors say for artesunate : » A drug that allows the late-stage parasites to develop normally may not be ideal…Merozoite release and reinvasion may exarcerbate disease symptoms and the subsequent immune response… ».The huge problem of resistance to artemisinin derivatives already discovered in 1990 may be due to this. Indeed, so far no scientific paper has described resistance to pure artemisinin, while numerous papers descibe the resistance to artemisinin derivatives and to ACTs, not only in Asian countries, but also in many countries of Africa and South America. As the authors further say : » The inhibitory action of artemisinin when added at the schizont stage is striking… a >600% increase in the number of unruptured schizonts ».

After rupture of the schizont the digestive vacuoles are selectively phagocytosed by leukocytes (PMNs). DVs generate also a lot of ROS and induce systemic inflammatory responses. It is the DV membrane more than hemozoin which causes these effects. All this leads to a significant reduction of the bactericidal capacity of the immune system and could explain why children affected with severe malaria frequently suffer from septicemia due to bacteria that otherwise play no major role in this potentially fatal affliction (P. Dasari et al., Blood, 2011 118-18, 49-60 & Dissertation zur Erlangung des Doktorgrades, Uni.Marburg 2013). The liberated DV after schizont rupture appears to function as a decoy and is exploited by the parasite to perturb central elements of the innate immune system and to divert PMNs from the merozoites . Despite the fact, as occurs in vivo, that merozoites outnumber DVs by an order of magnitude, PMNs preferentially contain DVs. Periodic attacks of fever in malaria are associated with schizont rupture that stimulates production of TNF-alpha. Overproduction of this endogenic pyrogen leads to severe and cerebral malaria. Artemisia plants have a strong immunostimulating effect. Infusions and capsules containing whole leaf powder might save thousands of children from coma and death.

All this may explain why very small quantities of Artemisia annua are sufficient to cure and stop the disease and why Bigpharma-WHO has to work with astronomic doses of artemisinin derivatives to achieve a similar result, but still be plagued with recurrence due to dormancy of parasites and sequestration caused by these massive doses.

The in vivo trials which have been run by our partners in Cameroon, Kenya, Congo, Burundi, Senegal, Central Africa, Uganda with Artemisia annua infusion or whole leaf tablets or capsules operate with artemininin doses far below 20mg/day and all give excellent results, even at 2mg/day. The bioavailabity of artemisinin is faster and four times higher if administered as tea (K Räth et al. Am J Trop Med Hyg, 2004, 70 :2, 128-132) and the therapeutic effect is even much higher if Artemisia annua is administered as whole leaf powder in lieu of pure artemisinin (M, Elfawal, P Weathers et al, PlosOne, Dec 2012, 7-12, e527446).

Quinine operates by inhibiting hemozoin formation, artemisinin derivatives do not (R Haynes et al., Antimicrob Ag Chemotherapy, Mar 2003, p 1175). The additional benefit of Artemisia tea infusion is that it also clearly shows this inhibitory effect. A mixture of Artemisia annua and Artemisia sieberi had a stronger inhibitory effect on beta-hematin formation than chloroquine, as demonstrated at the Al Quds university. (M Akkawi, P Lutgen et al., Brit J Pharm Toxicol, 2013, in press). Artemisia tea, or better even, whole leaf powder is a true polytherapy, containing dozens of antimalarial molecules, from essential oils to polyphenols, from coumarins to polysaccharides, from saponins to minerals. Plasmodium falciparum has very little chance to develop resistance simultaneously against them all.

It also has been demonstrated that regular consumption of Artemisia annua tea is boosting the immune system and has a strong prophylactic action (PE Ogwang et al., Brit J Pharm Res 1 :4, 124-132)


Pierre Lutgen