Isotope Labeling in Drug Development
Isotopes are atoms of the same chemical element that have different masses due to the presence of different numbers of neutrons in the nucleus, and there are light and heavy isotopes. According to physical properties, isotopes can be divided into two forms: radioactive and stable. Radioactive isotopes (e.g. 3H, 14C) undergo their own decay process and radiate radiant energy, are unstable and have a physical half-life. Stable isotopes are non-radioactive, physically stable, exist in nature in a certain proportion or abundance, are harmless to the human body, and can be labelled into drug molecules by chemical synthesis and tracked and detected by instruments such as gas and liquid.
- Radioactive Isotope Labelling
The widespread use of radioisotopes for active substance tracing relies primarily on two of their most important characteristics:
(1) Identity with the substance being traced, i.e. radionuclides and their non-radionuclides of the same element have a high degree of consistency in their chemical and biological behaviour, and do not disturb or disrupt the equilibrium of physiological processes inside and outside the body.
(2) Distinguishable from the substance being traced, the radionuclide's nucleus is constantly decaying, emitting rays that can be detected by a radioactivity detector, thus enabling the quantification and localisation of the marker.
In addition, the radioisotope tracer technology also has the advantages of high sensitivity, high exclusivity, wide applicability, and simple detection method, etc. Therefore, it has been widely used in drug ADME research, and the U.S. FDA has long taken the pharmacokinetic data of radioisotope-labelled drugs after drug administration as an important basis for the safety evaluation of new drugs and formulated the relevant guidelines.
The main advantage of stable isotope labelling reagents over radioisotope labelling reagents is that they are non-radioactive, do not require complex radiochemical equipment and radiation protection measures, and have no environmental pollution.
Appications of Isotope Labelling in Drug Development
Radioisotope-labelled compounds are more sensitive than stable isotope markers in quantitative analysis, and the measurement of radioactivity is not affected by non-radioactive impurities and chemical state, so the quantitative analysis is easier and the results are more accurate. In addition to solving the problems that cannot be solved by conventional analytical methods, radioisotope-labelled compounds can also be used as tracers to study the distribution, metabolism, efficacy and mechanism of action of drugs in vivo, which provides an important basis for pharmacokinetic study of drugs, and at the same time provides a methodology for the research and development of innovative drugs.
Potential toxicity study is a necessary part of drug discovery and clinical process. Previous toxicity studies mostly used in vitro experiments and animal experiments on a large number of compounds, which required a lot of money and time, and the mechanism of toxicity could not be explained. The use of stable isotope-labelled compounds allows us to trace the metabolism of drugs, identify the causes of toxicity and predict the potential toxicity of new compounds.
How to Choose an Isotope Tracer
Radioisotope Tracers
The commonly used radioisotopes in drug ADME studies include 14C, 3H, 32P, 33P, 35S, 125I, 131I, and so on. With the development of small PET instruments, the number of examples of ADME studies utilizing radionuclides such as 11C, 13N, 15O, 18F, etc. is increasing. The selection of radiotracer should be based on several comprehensive considerations such as experimental purpose, experimental period, and operator safety, including the ray type, half-life, radiochemical purity, specific activity, toxicity, and labeling position of the selected radioisotope. Commonly used radioactive tracers are mostly single radioisotope labeled compounds, sometimes for the special needs of the experiment, can also be used double labeled or multi-marked radioactive substances, but then the best choice of labeled atoms of different energies or emit different types of rays of the nuclide. The common ones are 14C/3H, 14C/125I, 125I/131I and so on.
Low-energy 14C and 3H are the two most commonly used radionuclides in the study of drug ADME, the half-life of these two nuclides are 5730 years and 12.35 years, respectively, due to their long half-life, the data measured in the experimental cycle generally do not need to be corrected for the physical half-life, which is convenient for the measurement and calculation of the results. Moreover, the beta-rays emitted by 14C and 3H are of low energy and easy to protect, which makes the experimental operation and result detection very convenient. In addition, 14C and 3H can also be detected by radiographic autoradiography with clear imaging.
Stable Isotope Tracers
Stable isotopes commonly used for tracing in the field of pharmacology are 2H, 13C, 15N, and 18O. Strictly speaking, stable isotopes refer to isotopes of an element that do not undergo or are very unlikely to undergo radioactive decay, e.g., 12C and 13C are stable isotopes of carbon, and in metabolism studies, stable isotopes are often used to indicate stable isotopes of relatively low natural abundance. Carbon exists in nature mainly in the form of 12C, with 13C accounting for only 1.11%, and nitrogen is dominated by 14N, with 15N accounting for only 0.37%.
Selection of Isotope Labeling Positions
Before preparing a labeled drug, it is first necessary to select a suitable isotope as the labeling element and decide on the location of the label. As a tracer study of the labeled drug, the labeled atom should be as representative as possible of the entire molecule of the drug in the organism, and the possible changes of the drug in the body should be taken into account. The most commonly used isotopes are 14C, 3H, and 35S, and sometimes 32P and 131I are also used. The isotopes used as the labeling atoms should be considered:
- The structure of the drug molecule
- The half-life of the isotope
- The energy of the radiation
- The ease of introduction of the labeling atom
- Special requirements of other tracer tests and other factors
As the most commonly used radionuclide, 14C is usually labeled on the skeleton structure of molecules, and its labeling site is more stable. 3H is easy to be exchanged with 1H in the surrounding environment, resulting in a decrease in the specific activity, so its stability is not as good as that of 14C. In addition, the isotope effect of 3H is more obvious than that of 14C, which limits its use to a certain extent. However, in general tracer experiments, the error caused by the isotope effect is often within the experimental allowable error, which is negligible. In addition, considering that the synthesis of 3H markers is relatively simple, 3H markers are often chosen as tracers when they can meet the experimental requirements.
In the selection of labeling sites, computer-assisted metabolite prediction technology (CAMP) can also be used to predict the stable sites in the molecular structure of compounds. In general, labeling of C atoms on aromatic or alicyclic rings in the molecular structure should be considered first, while labeling of active sites such as carboxyl, hydroxyl, sulfhydryl, amino, imino, etc. should be avoided as much as possible. This is because once these unstable groups are detached from the parent compound, the ability to trace the parent drug and major metabolites is lost. In addition, the labeling site should be far away from the location of chemical bond breakage to avoid the effects of isotope effects. If the parent compound generates two important metabolites at the same time due to chemical bond breakage during metabolism, double labeling technique can be considered when labeling the parent compound.
