‘Test medicines after five days’
What is the best method and time to test the effectiveness of medicines? PhD student Rob van Wijk of the Leiden Academic Centre for Drug Research (LACDR) discovered zebrafish larvae are best used in drug research when they are five days old. His findings were published on 15 February in the journal Scientific Reports and contribute to reducing, replacing and refining animal testing.
Internal or external
Drug developers increasingly use zebrafish larvae to test the effectiveness of new drugs. With these transparent larvae, they can easily and quickly study the effects under the microscope. Because the larvae are small, it is difficult to measure the concentration of medication in the blood. That is why researchers in zebrafish larvae use the concentration of medicine outside the larva. They dissolve medication in the water in which the larvae swim and a link the measured effect to the amount of dissolved medicine. 'The difficult thing is that you then still don't know how much medicine ends up in the larvae and actually creates the effect', says Van Wijk. Therefore, in collaboration with the Institute of Biology Leiden, he developed a method to determine this internal concentration using advanced equipment.
Development has impact
However, there was another factor to take into account. 'Larvae are by definition organisms in development. Just like in humans, this development affects the pharmacological processes that determine how much medication enters your body'. Therefore, in the second part of the research, Van Wijk determined the amount of paracetamol in larvae of three, four and five days old and compared these quantities. There appeared to be a considerable difference between them. 'The age of the larvae determines the exposure to medicine in the body.’ At the LACDR we have quantified this by means of mathematical models.'
Intake and breakdown
‘At the LACDR we then put our measurement data into mathematical models,' says Van Wijk. Two important results followed from these models. The first is that the medication intake of the larvae doubles between day 3 and 4. This makes the amount of medicine in a 4-day old larva twice as large as the day before. 'This is probably because the gastrointestinal tract opens between these two days', explains van Wijk. 'This creates an extra supply route so that medicine enters the body not only through the skin but also through the mouth.' Secondly, Van Wijk observed that the breakdown of medicines by the body increases by 17.5% per day. This also affects the internal concentration of medication. 'Because, if you apply the same dose, with a higher breakdown there will be less medicine left in the body.'
Side effects
Based on these two numbers, Van Wijk advises that the short-term effects of paracetamol are best investigated in five-day-old larvae. 'Then both the uptake of medication and the degradation are maximal.' The latter is relevant because the breakdown of medicine often releases secondary products, so-called metabolites. 'These metabolites can have either a toxic or a beneficial effect, and thus influence the effect of the drug. The chance that you observe these metabolites is greater if the degradation is greater.' Van Wijk chose paracetamol because this medicine has been studied many times already and therefore a lot of information was available. 'It would be great to be able to say on the basis of this one medicine: "we now understand them all". But unfortunately, that is not how it works. Every medicine is different. So we are now repeating our research with other medicines, such as an antibiotic and an anticancer drug.'
The three R's: reduce, refine and replace
By using the mathematical models at the LACDR, Van Wijk had to carry out fewer experiments to arrive at his results. 'Using models, you only have to perform a few experiments', he explains. The results are then used to predict the rest of the values. These virtual experiments allow us to obtain much more information from the available data, which also means that fewer experiments with larvae are required. In addition to reducing, Van Wijk's research also contributes to replacing and refining animal experiments. 'My research allows us to gain more information in the first stage of drug development, so you can design experiments for later/prosecutive studies more specifically. This way, experiments in larvae can replace follow-up studies in mammals and you can refine studies by designing them better.'