Tech ReviewTargeted Metabolomics in drug development
10.07.10Metabolomics systematically identifies and quantifies specific metabolites in a biological specimen. These metabolite data allow one to make inferences on the complex interactions between biological processes on a molecular level. Until recently, systems biology has mainly relied on three other ‘omics’ technologies: genomics, transcriptomics, and proteomics. Important as these areas have been, they fail to provide a real-time phenotype, i.e. a picture of what is actually happening in a biological system. Recent advances in mass spectrometry have added another powerful tool to the systems biology toolbox – metabolomics, or the study of the metabolome.
The metabolome is the sum of all low molecular weight metabolites in a biological system. By assessing thousands of metabolites simultaneously, modern mass-spectrometric techniques produce high-resolution biochemical snapshots showing the functional end-points of genetic predisposition, as well as the sum of all environmental influences, including nutrition, exercise, and medication. This snapshot is an almost real-time image of the physiology – or pathophysiology – of a cell or an entire organism.
The path to clinical applications
Mass-spectrometric assays revolutionised the diagnosis of inherited metabolic disorders. The development was co-pioneered in the late 1990’s by Biocrates’ founder Adelbert Roscher. This and similar pilot-projects around the world taught the diagnostics community some crucial lessons. First, quantitation of endogenous metabolites using multiple reaction monitoring (MRM) and stable isotope dilution (SID) on tandem mass spectrometers combined with advanced data analysis tools fulfils the most stringent quality criteria in terms of precision and accuracy, without suffering any of the shortcomings of immunoassays such as cross-reactivities. This makes the technology an ideal platform for clinical chemistry. Second, the theoretical assumption that multiparametric biomarkers would reduce biological noise in the data by internal normalisation and improve diagnostic sensitivity and specificity has been proven for many disorders. Third, this improved diagnostic performance was achieved without raising costs. To the contrary, neonatal screening is now reimbursed by health insurance providers in many Western countries, and it has led to substantial healthcare savings. These medical and commercial benefits turned neonatal screening into an impressive success story, and led to its introduction in most industrialised countries in less than a decade.
Targeted metabolomics vs. metabolic profiling
There are two approaches to metabolomics, and they are usually called targeted metabolomics and metabolic profiling. While both approaches are complementary, targeted metabolomics – the identification and quantitation of defined sets of structurally known and biochemically annotated metabolites – takes advantage of our detailed understanding of many biochemical pathways. Unlike protein-protein-interactions or regulatory relationships at the transcript level, the majority of essential enzymatic reactions are well characterised. In other words, the products and substrates of a reaction are known, as are its cofactors and often even its kinetics, equilibria, and energetics.
One major advantage of targeted metabolomics is that it generally provides quantitative information. These quantitative data – the molar concentrations of the metabolites involved in a pathway – facilitate the immediate understanding of any deviations from normal, and allow for comparison and meta-analysis of several independent studies. Interpretation of targeted metabolomics data is relatively straightforward regardless of which cohort is investigated – whether healthy or diseased, treated or untreated – because any alterations can be mapped to pathways in order to identify enzymes or pathways responsible for these changes.
Targeted metabolomics enables the systematic quantitation of a wide range of biologically relevant molecule classes in cells, tissues, or clinically relevant fluids. The technology comprises an automated sample preparation workflow integrated with sensitive mass-spectrometric methods and a tailor-made software solution. Many hundreds of metabolites can be identified and quantified using this novel platform, which is also well suited for high-throughput and routine applications.
Making the case – antidiabetic drug development
One of the most appealing advantages of metabolomics is that the majority of its analytes are not species-specific, i.e. most amino acids, sugars, lipids, etc. are structurally identical in mice, rats, dogs, pigs, monkeys, humans or other mammals, and even in most microbes. This makes metabolomics ideally suited as a biomarker platform that can be applied at all stages of drug development, without the need to re-develop analytical assays for every animal model and clinical trials.
One of the most striking examples for the value of metabolic characterisation of animal models was achieved in the early assessment of the efficacy of antidiabetic drug candidates. Specific phosphodiesterase IV inhibitors have anti-inflammatory properties and are used or are under development for indications such as chronic obstructive pulmonary disease (COPD) or asthma. When some researchers hypothesised that this drug class might also show anti-diabetic activity, we used targeted metabolomics to first characterise a widely-used mouse model of Type II diabetes, the db/db mouse, in unprecedented detail, and then assessed the efficacy of the lead compound compared to an approved drug (Rosiglitazone).
While previous studies were restricted to traditional end-points for antidiabetic activity such as blood and urine levels of glucose, we could quantitatively assess drug effects on many pathophysiological or pathobiochemical characteristics of type II diabetes. These range from insulin resistance of skeletal, muscle, liver and adipose tissue to oxidative stress, dysregulation of gluconeogenesis and glycolysis, urea cycle alterations or hyperphospholipidemia (Fig. 1).
In this context, the assessment of impaired short-term metabolic control and muscular insulin resistance turned out to be particularly telling. While the liver cannot metabolise the branched-chain amino acids (BCAA) leucine, isoleucine and valine, they are readily taken up by peripheral tissues like skeletal muscle and used as an alternative energy source. In the diabetic mice, there is a marked accumulation of the BCAA in peripheral blood because short-term metabolic control, i.e. the appropriate response to the availability of energy sources, is severely impaired (clear analogy to the lack of glucose uptake leading to hyperglycemia). In a study design with short-term treatment for only ten days, this marker yielded two extremely important findings:
– While Rosiglitazone could only slightly reduce the accumulation of BCAA, the candidate drug almost completely normalised BCAA levels and, thus, fully restored short-term metabolic control (Fig. 2).
– These dramatic metabolic improvements took place at a time when glucose as the typical end-point of such studies had not changed at all, i.e. such a biomarker is able to demonstrate pathology-specific efficacy much earlier or more sensitively than traditional clinical chemistry.
These and additional metabolic findings led to the decision to further develop this class of compounds as anti-diabetic agents, and the first substance is in successful Phase II trials at the moment.
Conclusions
Metabolomics, and particularly targeted quantitative metabolomics, has reached a degree of maturity that makes it amenable to a variety of applications, from basic research to clinical diagnostic to pharmaceutical R&D. Successful proof-of-concept studies demonstrated that metabolic biomarkers can serve as highly sensitive tools for characterizing animal models and assessing drug efficacy at a very early stage of drug development, thereby leading to evidence-based prioritisation of lead compounds. The same biomarkers have a significant potential to be used in clinical trials, to stratify patients and support therapeutic recommendations, and to monitor treatment efficacy much more effectively than conventional clinical chemistry.D
Contact
Klaus M. Weinberger, Ph.D.
Chief Scientific Officer
Biocrates Life Sciences AG
Innrain 66
A-6020 Innsbruck
Tel: +43-(0)512-579823
Fax: +43-(0)512-579823-4270
klaus.weinberger@biocrates.com
www.biocrates.com