Bioanalytical methods need to conform to lower volumes of blood required in efficacy and safety studies.
Reducing the number of animals used in drug development has always been the responsibility of those who actually work with animals, but the next big step in reduction and refinement will come not from toxicologists and biologists, but from bioanalytical chemists who often never even see a live animal.
I’m talking about microsampling, a valuable tool in helping us meet our 3Rs objectives (reduction, replacement, refinement). As the name suggests, microsampling uses a fraction of the blood that traditional methods do to assess whether a drug compound is safe and effective. Some companies are incorporating it into their workflows, but many others have not yet taken the steps to validate bioanalytical methods that conform to the lower volumes of blood required for microsampling in efficacy and safety studies. And that’s a problem.
Microsampling uses blood samples that are 70 uL or less than a typical blood sample volume of 0.5 mL (or 5000 uL). Microsamples are so small, in fact, that a typical safety study probably needs about a third to a half of the rodents delegated just for blood collection in traditional safety studies. In other words, microsampling has the potential of eliminating the need for hundreds of thousands of rodents, and refine and reduce the stress on large animal species as well.
But while a microsample is relatively easy to obtain, it is useless if the bioanalytical method to analyze the blood sample hasn’t been scaled down to require only microliter quantities of blood. Bioanalytical chemists have traditionally validated their samples using milliters of blood. With microsampling, they need to validate methods that use microliters of blood.
Choosing microliters over milliliters isn’t a question that can be settled mid-development, when you get set to do your blood draws in preclinical safety studies. Early on in the drug development cycle, usually even prior to the preclinical studies, drug developers determine the volume of blood required for any particular assay. Once a candidate drug is read for early-stage safety and efficacy studies, the bioanalytical methods using uL of blood that allow the measurement of the level of drug in the blood must be in place.
Current evaluations for exposure involve the averaging of three or more samples to obtain a mean average that is then compared to the toxicity observed in the other animals in the study. Being able to microsample the animals being evaluated for toxicity not only allows a better direct comparison of the toxicity being observed to a specific blood level, it allows us to see the variability in exposure from animal to animal within a sex and/or dose group.
As we move further into the drug development process, younger, smaller juvenile rodents may be used for testing and exposure has to be followed in these animals using separate animals that are used to measure the rate at which a substance gets into a body and what happens to it once it’s there. Initially, a rodent study may use 80 rats plus 54 rats for toxicokineticblood collection, this can change over time to 80 rats and 104 rats required for sampling of even younger rats. Juvenile studies currently require even more animals; over 150 animals for exposure analysis.
It should be clear that elimination of extra animals for exposure will reduce the over use of rodents in the drug development process by well over 50% and significantly reduce the stress of blood collection in large animals. However, without early consideration of microsampling techniques the use of microsampling will never become the norm, and our ability to reduce, refine and replace animals will plateau until assays totally replacing animals are available for routine safety testing. We know that this ultimate goal is still several years away, so until then there is real value in continuing to reduce and refine our techniques.