The importance of isotopic labelling in drug development: strategies and integration with biocatalysis and physical sciences

By Sean L. Kitson, Investigator of Radiochemistry at Almac

The author describes methods of delivering isotopically-labelled compounds suitable for use in clinical trials.

Isotopes utilised in drug discovery and development studies are capable of detecting associated metabolites formed in biological systems. These isotopes — most commonly, deuterium, carbon-13, tritium and carbon-14, are usually incorporated as late as possible in the synthesis to reduce timelines and cost.

Isotopes are incorporated into the drug molecule by several methods — the simplest approach being to exchange acidic protons within the drug using deuterated or tritiated water or hydrogenation with deuterium or tritium gas.  In some other cases, simple, commercial, labelled reagents, such as iodomethane can be used directly to generate the drug molecule.  These peripheral labelling approaches are susceptible to exchange with water or loss of the labelled group due to drug metabolism.  To circumvent these issues, carbon isotopes are often incorporated into the backbone of the molecule.  By using this approach, it is possible to prevent the loss of the label in a metabolically stable position during studies.

Isotopic labelling for ADME studies: investigating a drug’s absorption, distribution, metabolism, and excretion in the body

ADME studies are an essential part of drug development and involve investigating the drug’s Absorption, Distribution, Metabolism, and Excretion in the body. Incorporating specific isotopic labels into the investigational drug is a common approach used to carry out these studies. Both stable and radioactive isotopes can be used, but radioisotopes are more commonly utilised due to their easy detection and sensitivity.

A radiolabelled drug can be used in animal and human microdosing studies, and excretory products such as blood and urine are collected and analysed by several techniques to identify the in vivo metabolites. HPLC is a powerful technique used to analyse excretory products when radioactive labels are used, enabling the detection of most drug metabolites by the appearance of radioactive peaks other than those of the drug. Structural identification of the drug metabolites is confirmed using other techniques, such as multi-nuclear magnetic resonance and mass spectroscopy.

Common stable isotopes used to label investigational drugs include deuterium and carbon-13, while radioisotopes used include tritium and carbon-14. Tritium has a maximum specific activity of 29.1 Ci/mmol and an associated half-life of 12.3 years, while carbon-14 has a specific activity of 62.4 mCi/mmol and a half-life of 5,730 years.

The analysis of metabolites is a critical step in drug development and accurate quantification is essential. However, several factors can affect the quantitative measurement of metabolites in blood and urine, including ionic species and buffer ions that can enhance or suppress ionisation and analytical response signals, leading to distorted measurements. To account for these factors, stable labelled versions of the investigational drug are often required, in addition to radiolabelled versions. Carbon-13 labelled drug versions are typically preferred, as there is always a possibility of hydrogen exchange with deuterium.

After synthesising the isotopically labelled drug, it is necessary to accurately determine the isotopic enrichment by mass spectrometry. This process can be complicated when the compound in question contains a variety of labelled atoms, including carbon-13, nitrogen-15 and deuterium, or when the isotopes are radioactively labelled (e.g. carbon-14). Advances in time of flight (TOF) mass spectrometry have improved the resolution between the isotopes of labelled compounds, allowing for more accurate extraction of these isotopes than ever before. With high mass precision, it is possible to quantify the labelled composition of the compounds accurately.

Isotopic labelling of investigational drugs: importance of selective labelling and analytical methods for detection of radiolabelled molecules

The labelling of an investigational drug does not require every atom to be replaced and it may be sufficient to label only one position in the molecule. However, if a stable label version of the drug is required, the mass difference becomes important and it may require the incorporation of multiple isotopes, typically deuterium, nitrogen-15 and carbon-13.

Various analytical techniques can be used to detect radiolabelled molecules accurately, even if they represent only a tiny fraction of the total molecules present. This quantity is related to the molar activity (mCi/mmol) and represents the degree of radioactive labels incorporated into the drug under investigation. Knowing the molar activity of the starting material is advantageous as it can be used to follow the radiochemical assay of the intermediates throughout a radiolabelled synthesis.

However, isotopic labelling of modern APIs, particularly complex targeted therapies, is becoming more challenging. The synthetic design of an isotopic analogue depends on several factors that must be carefully considered, along with any prior knowledge of the molecule, to ensure that a metabolically stable position is labelled.

The availability of appropriate labelled starting materials is limited and the high costs of carbon-14 reagents mean that these costs must be carefully weighed against labour costs for a longer synthesis.

Integrating isotope chemistry with biocatalysis and other technologies: providing innovative synthesis options for isotopically-labelled compounds

Actively integrating isotope chemistry with other in-house technologies can allow you to find fit-for-purpose solutions beyond the traditional realms of chemical synthesis. Biocatalytic tools, such as enzymes, introduce complex functionality in a single step and significantly help reduce the cost of labelled biomolecules and chiral compounds.

Biocatalysis and isotope chemistry integration

In our experience at Almac, the benefits of linking our isotope chemists with our experts in biocatalysis, physical sciences and peptide protein technology (PPT) groups have been innumerable. This integration affords us new and attractive synthesis options and shorter synthetic routes. Furthermore, it ensures that best-in-class technical solutions can be found to the many challenges that we faced during the synthesis of isotopically labelled analogues.

The integration of isotope chemistry and the other technologies mentioned above facilitates the synthesis of isotopically-labelled peptides and bioconjugates, as well as small molecules to GMP standards. This results in the delivery of isotopically-labelled compounds suitable for use in clinical trials.


*Dr Kitson is an investigator at Almac and has more than 22 years of experience in the synthesis of carbon-14 radiolabelled compounds. He is also the former Editor-in-Chief of Current Radiopharmaceuticals and a scientific committee member of the International Isotope Society (UK Group). He is a recipient of the 2006 Wiley Journal of Labeled Compounds and Radiopharmaceuticals award for radiochemistry.