Are in vitro antimalarial assays a waste of time?
In vitro efficiency does not guarantee in vivo
Most of the assessments on the antimalarial efficacy of a molecule are made in vitro. In the case of plant material, the first step is generally the extraction with an organic solvent. The extract is then lyophilized, frozen and stored for subsequent trials. A recent research paper from the University of Al Quds Akkawi M, Jaber S, Abu-Remeleh Q, Engeu OP, Lutgen P (2014) . Investigations of Artemisia Annua and Artemisia sieberi Water Extracts Inhibitory Effects on β-Hematin Formation. Med Aromat Plants 3: 150. doi: 10.4172/2167-0412.1000150) showed that lyophilized extracts of an aqueous Artemisia infusion lost activity with time, which may have an impact not only on in vitro laboratory results but also in vivo treatment efficiency obtained with old extracts or molecules contained within. Nevertheless, stability of these extracts is much greater than that of arteminisinin, artesunate and dihydroartemisinin, in vitro, ex vivo and in vivo. A stability test at 37°C in a medium supplemented with 10% inactivated fetal calf serum gave half-life values of 10.3 h for artesunate, 5.2 h for dihydroartemisinin and 11.2 h for artemisinin. Alyssa Flobinus, Nicolas Taudon, Marc Desbordes, Beatrice Labrosse, Francois Simon, Marie-Christine Mazeron and Nathalie Schnepf, Stability and antiviral activity against human cytomegalovirus of artemisinin derivatives. J Antimicro Chemotherap doi:10.1093/jac/dkt346 Furthermore, many extracts are obtained with organic solvents and miss the molecules contained in a plant which are only water soluble, like polysaccharides or condensed tannins or minerals. And when water is used it is ultrapure water which has nothing to do with the tap or well water the infected person is going to use for the ingestion; pH and ionic strength of the water have an effect on solubility and pharmacodynamics. The extracts are generally passed through filters with ultrafine pore sizes, which will remove all kinds of bulky molecules like polysaccharides, tannins or their aggregates. In the next step, most research efforts will try to fractionate the extract in its individual molecules to assess the efficacy of each one isolated. It is unfortunate that in vivo results obtained with these isolated molecules will lead to monotherapies as it is the case with artemisinin and its derivatives. It does not make much sense to add a second molecule like amodiaquine which has already shown severe signs of resistance since more than twenty years. It is putting two leaking buckets on into the other. Artemisinin Combined Therapies or ACTs well designed booby trap for Africans. A growing body of studies, especially those of the Worcester Polytechnic Institute Mostafa A. Elfawal, Melissa J. Towler, Nicholas G. Reich, Douglas Golenbock, Pamela J. Weathers, Stephen M. Rich. Dried Whole Plant Artemisia annua as an Antimalarial Therapy, PLOS. Dec 20, 2012 http://dx.doi.org/10.1371/journal.pone.0052746) have shown that compared to pure artemisinin, delivery of the drug via oral consumption of dried Artemisia annua leaves better fights parasitemia, delivers far more of the drug into the blood, persists longer in the blood of infected animals than healthy ones, requires far less of the drug to get a better therapeutic effect, and is safe and effective in human malaria patients. Furthermore, the dried plant offers a plethora of other endogenous chemicals that could well thwart emergence of drug resistance; many show some anti-plasmodial efficacy. Some work in concert with artemisinin. There are claims that use of tea or dried leaves is just monotherapy and will cause artemisinin drug resistance. But these claims never have been assessed by clinical trials…thus nobody knows. It is crucial that decisions affecting millions of lives be made on solid scientific facts and not just on fearful conjecture. The debate on in vitro versus in vivo is linked to the debate on efficacy versus effectivenes. Ideally one can only talk about efficacy in laboratory conditions because the experiment can be fully controlled. Measuring plasma levels of drug alone is not sufficient for efficacy because many other factors such as food, immune status, environment, genetics etc influence drug efficacy. Therefor effectiveness describes the real-life situation, but the problem is that mathematically we cannot compute real life situations and this is why most studies compute and report efficacy by having treatment group versus control group data compared. Even then inter human and intra human variations cannot be completely controlled. When consumed consciously and systematically, many wild plants are very important for human health because of their constituents. The effect of essential oils of plants on microorganisms in vivo cannot be predicted from in vitro assays as concluded by the paper hereafter. Sevil Toroglu. In vitro antimicrobial activity and antagonistic effect of essential oils from plant species. Journal of Environmental Biology July 2007, 28(3) 551-559 (2007 Even more, in vitro trials may show the absence of an effect which is well present in vivo. The University of Leiden found that Artemisia afra aqueous infusion has no in vitro antimalarial effect, but numerous clinical trials in Africa show that it has a strong effect. J Mouton, O Jansen M Frédérich, Fr van der Kooy, Is artemisinin the only antiplasmodial compound in the Artemisia annua tea infusion. Planta Med 2013, 79, 468-470. Some compounds exist in what we call pro-drugs, they need to be activated by some enzyme system, in order to become active. In vitro tests miss such drugs because they lack the enzyme system or related factor. Actually, most plant medicines have been wrongly lost by relying on in-vitro systems when they show negative results. Also, some compounds may be active in vitro but have zero effect in vivo because the body may have enzyme system that destroys it before it produces effect. A human body is complex and no one understands it fully. Even if in vitro you have a fantastic efficacy, you may have a negligible efficiency in vivo. Or often the reverse: you may have zero efficacy in vitro but still a high in vivo efficacy. This complexity is evident in the case of flavonoids where the effects have all been studied in vitro. The in vivo administration of purified forms of these compounds often fails to show an effect. Kaempferol administered to rats did not inhibit CYP3A4. Li P, Callery PS, Gan LS, Balani SK. Esterase inhibition by grapefruit juice flavonoids leading to a new drug interaction. Drug Metab Dispos. 2007 Jul;35(7):1203-8. Epub 2007 Apr 23. The in vitro inhibitors naringin and quercetin do not contribute to the in vivo inhibition of CYP3A4 either. Quercetin, an in vitro inhibitor of CYP3A, does not contribute to the interaction between nifedipine and grapefruit juice. Rashid J, McKinstry C, Renwick AG, Dirnhuber M, Waller DG, George CF.Br J Clin Pharmacol. 1993 Nov;36(5):460-3. Scopoletin in vivo even displays an antimalarial activity which had never been detected in vitro The distribution of scopoletin in murine tissues after oral administration also was studied in China Yu-feng Xia, Yue Dai, Qing-yu Meng, Qiang Wang, Ling-ling Qiu. Studies on absorption kinetics of scopoletin in rat stomachs and intestines. Zhongguo Zhong Yao Za Zhi 2008 Aug;33(15):1890-4 Some drugs are just designed to kill the parasites released into the blood stream, but they leave a bloody battlefield and a depressed immune system. Repeated malaria attack results into excess haemozoin produced by parasites when they digest the haemoglobin for their food. Accumulation of haemozoin in blood weakens the white blood cells that help in mopping out of malaria parasites after treatment. There is no drug that eliminates all the infections from the body. Drugs help to reduce the parasite load and our immune system does the mop up. So, if the immune system is weak, the residual parasites are not removed and the infection surges again causing malaria. A thesis from the Virginia Polytechnic Institute Jill M. Squires. The Effects of Naturally Occurring Plant Products on Experimental Haemonchus contortus Infection in Gerbils and Sheep. Thesis submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Master of Science, 2009 studying the effects of plant products on Haemonchus contortus in gerbils finds that artemisinin treated groups exhibited higher main parasite burden than control groups. Artemisia annua extracts however produce a noticeable reduction in parasite load. The authors conclude that other substances present in Artemisia might cause this beneficial effect, and they demonstrate that limonene has an extraordinary efficacy. Patent WO2006120568, when comparing the antimalarial of artesunate and carvacrol in vivo on Plasmodium berghei infected mice find that artesunate does not protect the animals, that carvacrol alone has a remarkable antimalarial activity, but that the two are synergistic. A more recent work Trifone D’Addabbo,Teresa Carbonara, Maria Pia Argentieri, Vincenzo Radicci, Paola Leonetti, Luciano Villanova, Pinarosa Avato. Nematicidal potential of Artemisia annua and its main metabolites. European Journal of Plant Pathology. October 2013, Volume 137, Issue 2, pp 295–304 finds that the nematocidal efficiency of Artemisia aqueous extracts was high, but that the efficacy of artesunate was zero and that of artemisinin low. The same applies for cancer therapy. During many years, artemisinin and its derivatives artesunate and artemether were proclaimed as new wonder cure against cancer. But the cytotoxity of artemisinin against a series of cancer cells is in no way stronger than that of other molecules present in Artemisia annua, like scopoletin, arteannuin-B, artemisitene, 1.8-cineole ; and artesunate has led to severe resistance of some cancer cells. A death sentence! Efferth T, Herrmann F, Tahrani A, Wink M. Cytotoxic activity of secondary metabolites derived from Artemisia annua L. towards cancer cells in comparison to its designated active constituent artemisinin. Phytomedicine. 2011 Aug 15;18(11):959-69. doi: 10.1016/j.phymed.2011.06.008. When comparing the in vitro effects of aqueous extract of Artemisia sieberi and artemisinin on Leishmania major It was found that the aqueous extract was stronger than pure artemisinin and completely eliminated the promastigotes. Esavan Heydari F, G Affarifar F, Soflaei S, Dalimi A. Comparison between in-vitro effect of Artemisia sieberi and artemisinin on Leishmania major. Jundishapur J Nat Pharm Prod. 2013; 8(2): 70-75. Ean-Jeong Seo, Victor Kuete, Onat Kadioglu, Benjamin Krusche, Sven Schröder, Henry Johannes Greten, Joachim Arend, Ik-Soo Lee, and Thomas Efferth. Antiangiogenic Activity and Pharmacogenomics of Medicinal Plants from Traditional Korean Medicine Evid Based Complement Alternat Med. 2013; 2013: 131306. Published online 2013 Jul 22. doi: 10.1155/2013/131306 Our own in vitro trials show an interesting antitumour effect against HeLa, BG, Fem-x, LS, MDBA-MB-361, K562 cells without significant cytotoxic effect against PBMC cells. But that does not mean that the same effect will work in vivo. In the absence of clinical trials of Artemisia plants against cancer caution is required and the numerous advertisments on internet that Artemisia annua cures cancer in a few hours or days are irresponsible Z Juranic, I Matic, P Lutgen, In vitro study of the antitumor effect of Artemisia annua tea. EJC Supplements, 2010 8,5, 81 A more extensive study by the Swiss Tropical Institute shows that artemisinin is active against Plasmodium falciparum in the ng/ml range but against Trypanosoma brucei, Trypanosomi cruzi, Leishmania donovani, Giardia duodenalis, Babesia divergens the activity is 3 orders of magnitude lower. Kaiser M, Wittlin S, Nehrbass-Stuedli A, Dong Y, Wang X, Hemphill A, Matile H, Brun R, Vennerstrom JL. Peroxide bond-dependent antiplasmodial specificity of artemisinin and OZ277 (RBx11160). Antitimicrob Agents Chemother. 2007 Aug;51(8):2991-3. In vitro prooxidants like hypochlorite, hydrogen peroxide, artemisinin are effective in the micromolar range, but in vivo much higher concentrations are required because of the rapid metabolism. And we should not forget that based on the position of WHO/MAL/98.1086 for in vivo treatment of malaria doses are prescribed which are at least 4 orders of magnitude higher than the 1-2 ng/ml values found in in vitro. Nevertheless, the problems of failure and resistance become overwhelming in Asia, Africa and South America. ACTs are the combination of two failing monotherapies, Artemisia annuais a true polytherapy. Artemisia annua tea or powder, even when low in artemisinin, gives a cure rate >95% and never has shown any sign of resistance. The question may even be asked if artemisinin is as efficient against malaria in vivo as in vitro.