Abstract
Biography: Maria Veneziano is currently a Research Investigator in the DMPK unit at IRBM (Pomezia, Italy), an Italian CRO and biotech company specializing in preclinical drug discovery of small molecules, peptides and antibodies. She studied Biological Science at ‘Federico II’ University of Naples (Italy) and completed her PhD in Medical Biotechnologies at Merck Research Laboratories (MRL) in Rome (Italy) developing bioanalytical methods used to identify and quantify amino acids and acylcarnitines for the diagnosis and follow-up of inborn errors of metabolism. As part of the DMPK team at MRL, she was involved in PK and ADME profiling of small molecule and peptide candidates for drug-discovery programs. Presently, Maria leads a group supporting PK and PK/PD studies for small molecules and peptides.
Maria Veneziano speaks to the International Journal of Pharmacokinetics about her experience working on pharmacokinetic studies. She starts by discussing the conventional bioanalytical methods used for the quantitative analysis of small molecules and peptides and she highlights the important role of LC–MS detection and sample preparation in the bioanalysis of pharmacokinetic studies. She also speaks about the role of high-resolution mass spectrometry in the bioanalysis of peptides as an important tool in a drug-discovery program to simultaneously define pharmacokinetic and metabolic profiles of the same drug candidate. She also describes cassette dosing and cassette analysis approaches as strategies to increase sample throughput, highlighting advantages and limits of each of these strategies. Finally, Maria speaks about her idea of ‘simplified PK workflow’ based on the miniaturization and automation of all the steps in a PK study, from in vivo administration to sample analysis.
Please introduce yourself & your experience working on pharmacokinetic studies
My name is Maria Veneziano and I am currently a Research Investigator in the DMPK Unit at IRBM; as a DMPK scientist with a decade of experience within the pharmaceutical industry, my main research interest is pharmacokinetics and its application to drug discovery and development programs. During my career, I gained extensive experience in the design, conduction and interpretation of PK studies of small molecule and peptide drug candidates. With over 15 years experience in quantitative LC–MS analyses of small molecules and peptides in biological matrices (e.g., fluids, dried blood spot and tissues), I implemented new technologies for high-throughput PK studies such as online extraction by turbulent flow chromatography. I have also contributed to the improvement of the PK process efficiency by the integration of microsampling, from in vivo administration to sample preparation and analysis, and using my proficiency with Watson LIMS for automating data handling and reporting tasks.
Can you briefly summarize the conventional bioanalytical methods used for pharmacokinetic studies?
The two main components of a bioanalytical method are the sample preparation and the compound detection. The most commonly used sample preparation techniques for PK studies are plasma protein precipitation, solid-phase extraction (SPE) and liquid–liquid extraction. Due to the simple and fast clean-up of samples, the protein precipitation is the most employed technique for extraction of compounds from biological matrices. However, for a selective removal of matrix interferences we typically use SPE approaches which are increasingly adopted due to the high recovery of the new devices and their integration in the robot sample preparations. An alternative and powerful approach for on-line extraction in bioanalytical studies is the turbulent flow chromatography. This technology improves sensitivity and reduces sample preparation time, two factors that are of primary importance in drug discovery. Currently, due to its sensitivity and selectivity, LC–MS is the principle technique used in quantitative bioanalysis and the triple quadrupole mass spectrometer is the instrument of choice for PK study samples analysis. However, the latest generation of high-resolution mass spectrometer systems are evolving to offer an efficient alternative for quantitative analysis of compounds in biological matrices. LC–MS has also emerged as a promising alternative or a complementary technology to compensate for the lack of specificity of traditional approaches adopted in the bioanalysis of macromolecule drugs (proteins and antibodies) such as ligand-binding assays.
What are the key challenges in the development of the bioanalytical methods for pharmacokinetic study sample analysis?
Due to very demanding requirements in terms of method reliability, sensitivity, speed of analysis and sample throughput, the development of bioanalytical methods supporting pharmacokinetic studies has become more and more challenging in recent years. These requirements affect the choice of sample preparation techniques and instrument platforms. One of the main steps in the development of a bioanalytical method is selecting a highly sensitive LC–MS method to detect and quantify low drug concentrations and to accurately determine PK parameters of the compound drug candidate. This is a very challenging step especially for bioanalysis supporting microdosing PK studies due to the low doses administered and the very low drug concentrations (from ng/ml to pg/ml) associated. A preconcentration of sample is often adopted to increase the sensitivity even if this approach increases not only the target analyte concentration but also the interferences of the sample matrix – one of the major problems for analysis of biological samples using LC–MS. The interferences from the sample matrix may affect the ionization of the target analyte with the consequence of ion-suppression or enhancement in the LC–MS response that may dramatically compromise the analysis. Based on experience, the choice of a highly selective sample preparation for an efficient sample clean-up is mandatory to develop an appropriate bioanalytical method for PK study samples analysis.
What is the role of high-resolution mass spectrometry in performing bioanalysis of pharmacokinetic samples?
Due to the selectivity and sensitivity performance, triple quadrupole mass spectrometry system represents the method of choice of analytical scientists to quantify the parent drug, drug metabolites or biomarkers in biological matrices. However, a worthwhile alternative for the bioanalysis of small molecules and peptides is the new generation high-resolution mass spectrometers (TOF and Orbitrap) that offer good performance for quantitative analyses, in terms of sensitivity, dynamic range, resolution and accuracy. In our laboratory, the use of high-resolution mass spectrometry (HRMS) is the preferred option for bioanalysis of peptides especially for those that are hard to fragment. Unfavorable fragmentation may hamper the identification of sensitive and selective multiple reaction monitoring transitions but, thanks to the ability to perform a quantitation based on precursor ion-chromatograms in full‐scan MS with an advanced sensitivity, HRMS is an alternative and more effective approach for dealing with difficult peptides. The advantage of using HRMS to analyze the sum of some or all of the multiple ionic species of the peptides is the improved sensitivity and robustness of the analysis. As an important tool at the early stages of a drug discovery program, HRMS in full-scan mode offers the possibility to obtain, in the same run, pharmacokinetic and metabolic profiles of a compound drug candidate.
What are the approaches for increasing sample throughput in the pharmacokinetic studies?
To meet the requirements of an increasing throughput of PK studies, the DMPK scientists routinely adopt strategies, such as cassette dosing and cassette analysis, to reduce the number of samples to be analyzed. With cassette dosing, multiple compounds are dosed simultaneously to the same animals. This approach reduces the number of test animals, providing a complete PK profile for each compound in individual animals. However, potential in vivo drug–drug interaction limits the doses we can use. Furthermore, a careful selection of the compounds is essential to assess multiple compounds together and requires a more complex bioanalysis due to the presence of multiple analytes, potential interferences and signal suppression from pooled compounds or metabolites. The alternative strategy of cassette analysis is referred to as ‘pooling by time’ or ‘pooling by compound’. With ‘pooling by time’, the same time points from different studies, involving multiple compounds, are pooled. As a consequence, the overall time for analysis is decreased but the sensitivity is also decreased due to sample dilution after pooling. Therefore, a more complex bioanalysis is required to select a highly sensitive and selective LC–MS method as well as an appropriate sample preparation. With ‘pooling by compound’ the samples from the same time points of the two or three animals dosed in a single study are pooled into one single sample; this approach provides all PK parameters saving both sample preparation and analysis time but it does not provide any information about the variability in PK parameters among animals.
What are the most common challenges of pharmacokinetic studies in modern-day laboratories & what solutions do you use to address these?
One of the most important aims in PK studies is the reduction of the sample volumes collected from animals (microsampling), which in turn leads to a reduction of animals used for PK studies. Over recent years, our work for the implementation and refinement of the capillary microsampling technical approach (CMS) in preclinical PK studies has had a positive impact on the ethical and financial aspects of our laboratory. Not only has microsampling contributed to the reduction of animal numbers and consequently to the cost associated with a PK study but it has also allowed for the introduction of less invasive sampling procedures with blood collection taking place in a glass capillary micropipette from the tail vein of the rodent. Furthermore, serial blood collection from the same animal has had a beneficial impact on PK data quality. Understandably, the use of ‘microvolumes’ has required both the implementation of LC–MS method and the optimization of the automated sample preparation to manage ‘microsamples’. At present, the DMPK laboratory at IRBM can manage 10 μl matrix sample volume in the sample preparation but we are working to reduce this volume further. The rationale behind this further reduction is to enable the samples collected from the same animal to be split into several aliquots which can then be used for multiple analysis, for example, PK and PD sample analysis from the same study, with a subsequent improvement of data interpretation and correlation.
What emerging technologies are you particularly excited about, which are likely to change the future of pharmacokinetic studies?
Miniaturization is a new challenge for PK studies. The replacement of conventional approaches by miniaturized alternatives offers advantages scientifically and ethically. For example, sampling and sample preparation are the first steps encountered in bioanalysis of PK studies and are crucial to ensure appropriate biological interpretation. Microsampling and microextraction techniques have been developed in our laboratory to enable handling of low volumes samples. These technical approaches combined with simplified experimental operations and reduced time of analysis have translated into a miniaturized bioanalytical/PK workflow that must be the new trend in PK studies. As previously discussed, the reduced blood volumes taken from animals by microsampling enable serial sampling from the same animal rather than composite bleeds from several animals. As a consequence, the exposure and potential toxic effects of a drug compound can be assessed within the same animal with a better quality of PK data. However, for managing low doses/volumes, significant efforts are required in analytical method optimization to reach the desired levels of sensitivity as well as in the development of automated sample preparations (liquid handling systems, extraction devices and procedures, laboratory materials). The use of modern technologies in our laboratory, such as advanced liquid handling systems for sample preparation combined with sensitive and selective mass spectrometry systems has allowed us to build up a simplified bioanalytical/PK workflow with significant improvements in terms of data quality and increased sample throughput.
Disclaimer
The opinions expressed in this interview are those of the interviewee and do not necessarily reflect the views of Newlands Press Ltd.
Financial & competing interests disclosure
M. Veneziano is an IRBM employee and the company has a financial interest with the subject matter and materials discussed in the manuscript. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.