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Are in vitro antimalarial assays a waste of time?

March 22, 2014 - 08:59 -- Pierre Lutgen

by Patrick Engeu Ogwang and Pierre Lutgen

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 (M Akkawi et al., Med Aromat Plants, 2014, 3:1) showed that lyophilized extracts 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.

Furthermore, these extracts obtained with organic solvents miss the molecules contained in a plant which are only water soluble, like polysaccharides. 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 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. A well designed booby trap for Africans.

A growing body of studies, especially those of P Weathers (M A Elfawal et al., Plos One, Dec 2012, 7:12, e52746) 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.

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 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.

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 ones 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.