In Part 1, Dr Johanna Haglund (Head of Safety Metabolism) described the first steps to be taken in the Metabolites in Safety Testing evaluation (MIST) and in this second part, she will elaborate how to proceed after in vitro cross species comparison between human and safety species.
In vivo MetID in toxicology species, why and when?
How do you evaluate the in vitro cross-species data?
The in vitro data often reveal the metabolic soft spots and major metabolic pathways of a drug candidate. However, it is still important to keep in mind that in vivo data gives a more reliable picture, since there might be sequential metabolism involved, which cannot always be captured in vitro. Although in vitro metabolism is the best option to get a first glance on hepatic metabolism, the quantitative and qualitative discrepancies are often related to lack of enzymes or pathways. In addition, in vitro environment is a static one compartment system compared to the body where metabolism occurs in a dynamic environment with several compartments and is dependent on transporting etc. Therefore, the in vitro data might demonstrate human metabolites, which are not formed similarly in the safety species. However, it does not necessarily mean it is unique to human, but it is human specific under the in vitro test conditions.
What would be the next step after in vitro cross-species comparison in terms of MIST evaluation?
According to our experience and for the reasons mentioned above, it is extremely valuable to further investigate/confirm in vitro data by performing in vivo MetID in safety species, using any available in vivo samples. Although human metabolite profile is the most interesting, in vivo safety species analysis helps to confirm potential risk for in vitro/in vivo discrepancies and to answer the questions; How relevant were the in vitro profiles?; What can be further expected in vivo also in human?; Is multi sequential metabolism involved?; Have all the metabolic pathways been identified in vitro?; Was the lack of certain in vitro human metabolites in safety species only an in vitro phenomenon or is there a true absence also in vivo in safety species?
How do you choose species to be included for the in vivo MetID?
If available, I would choose both general toxicity species to start with. Either at the same time or start with first tox species (rodent) and if considered important, continue with second tox species (non-rodent) depending on the outcome of the in vitro cross-species profiling. The more you learn about the metabolic faith of the drug candidate, the better you are prepared for further considerations (DDI, excretion, etc).
What kind of samples to use in the initial in vivo MetID?
At this point it is not critical. I would use the most relevant samples available at this stage. It could be samples from pre-nomination tox, DRF, MTD or GLP tox. The aim here is to understand what metabolites are formed and circulated after in vivo administration and the results are used to build the metabolic pathway of the candidate drug Of course, one must consider that a high dose in MTD study might not be relevant in quantitative terms. Still, it provides information on metabolic pathways if these samples are the only ones available.
What could be the possible outcomes from in vivo MetID?
The data obtained with in vivo samples may bring in several new points to consider, which have not been revealed earlier. You might realise direct glucuronidation is an important step in animals, which is not seen at all or not as pronounced in vitro and can be expected to take place in human as well. You might detect several intermediates in a sequential metabolism which are not detected in vitro, as is the case with piperazine. You might also find glutathione adducts or derivatives thereof that normally are not seen in vitro. You might find out, that the lack of certain human in vitro metabolites in the toxicity species was only an in vitro phenomenon, arising from lack of enzymes or less turnover.
In conclusion, you learn a lot on the fate of the molecule. Now the most important step is to perform MetID in vivo in human to establish the in vivo human metabolite profile. This allows you to understand which metabolites might be of concern and if they were seen in the tox species in vivo?
Written by Johanna Haglund