Despite a good part of ADME research in drug discovery and preclinical development can be performed using various in silico or in vitro systems, eventually it becomes necessary to evaluate also the pharmacokinetic (PK) profile in animals to elucidate in vivo DMPK properties of the drug candidates. This blog post summarises the components of a typical early PK study.
Mice and rats are often used in the first PK studies to get initial understanding about the compounds’ PK characteristics. The information may be used to select most promising candidates for further studies or on the other hand, the results may reveal a need of going back to the chemistry lab for optimising the molecules to improve their performance or the first PK results may even put an end to a promising project.
Typically, the compound is dosed perorally and intravenously, which allows determining oral bioavailability – a crucial information for a drug candidate intended for peroral administration. Also, the development of preclinical formulation plays a big role in a PK study, since it may affect the PK profile of the compound.
Usually the compound is administered first as a single dose. This is followed by collection of blood samples up to 24 hours. The collected blood samples are centrifuged, and the obtained plasma samples are frozen for storage until analysis. Refinement methods, such as microsampling, and use of sensitive analytical techniques allow collection of low blood volumes, which also reduces the number of animals needed. In addition to single dose studies, repeated dose studies may be performed, where the compound is administered repeatedly over couple of weeks. Later, in more advanced studies and to answer more specific research questions, bile duct can be cannulated to evaluate biliary excretion and use of metabolic cages enables urine and faeces collection for analysing the eliminated compound or metabolites. In addition, various tissues can be collected, and their drug concentrations analysed to have information about tissue distribution.
Bioanalysis is a critical part of the PK study to have reliable data about the drug plasma or tissue concentrations. Typically, the samples are analysed with LC/MS. An important step prior LC/MS analysis is sample preparation to generate a final sample, which is suitable and optimised for LC/MS analysis – a topic worth of its own blog post! The benefit of using LC/HR-MS is the possibility to also mine and identify the drug metabolites from the same data.
After the plasma samples have been quantified for the compounds, its time for the scientist to sit down and process the data. Typically, a specific software for PK analysis is used and a non-compartmental model applied, which requires reliable estimation of total drug exposure. Shortly put, the total drug exposure is estimated with area under the plasma concentration–time curve (AUC) with the trapezoidal rule. The most interesting PK parameters at this stage are peak plasma concentration (Cmax) and the time when it is observed (Tmax), clearance, half-life and especially bioavailability. An experienced PK scientist can interpret the results and help to understand their meaning.
To summarise, before the PK data is available for guiding the next steps of the project, there are several tasks to be conducted. Luckily, there are experienced high-quality CROs that can offer guidance and support through all the steps – starting from study planning and formulation all the way to the in vivo work, data analysis and interpretation and reporting. Eventually, each project is unique, and it may be necessary to design a specific experimental set up. That is why input from experienced PK scientists and chemists is highly valuable.
Use of animals in research is strictly regulated by the European and national legislation. Approval from the authorities is required for conducting any research using animals. Animal treatment should be responsible and the 3Rs (replacement, refinement and reduction) applied to cover all the aspects, such as housing, animal care and use. Besides to improved animal well-being, responsible use of animals optimises the reliability of the obtained data. There are also additional, voluntary ways to further improve humane use of animals in research. AAALAC International is a private, independent organisation, which promotes animal wellbeing and evaluates organisations for the use of animals in research and accreditation is awarded if the standards are met or exceeded.
If you wish to read more about in vivo PK studies, why wouldn’t you check out our e-book on the topic?
Written by Miia Kovalainen
