For certain DMPK studies the availability of a radiolabelled form of a test item is highly desirable. 

 

Distribution and mass balance can be readily assessed and crucially, metabolites can be easily quantified without the need for bioanalyical standards.

 

But when considering a radiosynthesis, there are some key issues which should be considered:

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+ Choice of Radiolabel

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Typically for metabolism studies of ‘small molecule drugs’ or new chemical entities (NCEs), the radiolabel will be carbon-14 (14C) because it has a suitably long half-life (5730 years) and is less subject to ‘exchange issues’ – the exchange of a radioactive atom for a non-radioactive atom – which can be observed with tritium. However, the use of tritiated (3H) compounds should not be overlooked, particularly for discovery and lead optimisation studies, where often lower amounts of compound are available and financial resources are more limited.

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Modern tritiation methods (site-directed catalysis) can now direct the location of insertion of the tritium atom, ensuring the molecule is radiolabelled in a metabolically stable position. Tritiated compounds can be used for a similar range of metabolic investigations as those labelled with 14C.

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Other radioisotopes may be used to label specific compounds, exploiting their unique chemical properties. Drug candidates labelled with iron-59, sulphur-35 and zinc-65, among others have been used for metabolism studies. Iodine-125 has been a popular radioisotope for labelling peptides and proteins, enabling the biodistribution of biological drugs to be followed. However there are disadvantages with working with a gamma emitter and other methods to radiolabel peptides and proteins have been developed, e.g. covalently binding a radiolabelled 'tag' to the molecule.

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+ Position of Radioisotope Labelling

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One of the important aspects of custom radiolabelling is to try and ensure the radioisotope is incorporated in the molecule in a metabolically stable position.

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That is, incorporate the radioactive atom in a position in which the atom is retained following metabolism so that it is present in the metabolites. The metabolites can then be monitored and quantified by radio-HPLC.

 

To help assess the potential major metabolic liabilities of a compound prior to initiating custom radiosynthesis, initial in vitro metabolism studies using a non-radiolabelled form of the compound, with analysis by LC-MS/MS is often useful.

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+ Specific Activity

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Specific activity relates to the radioactive activity (measured in Curies, Ci or Megabequerels, MBq) per unit mass of the compound synthesised – via the incorporation of radioactive C or H atom. In order to make comparisons between compounds it is important to use molar specific activity.

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Generally, the higher the molar specific activity the better, assuming radio-instability is not an issue. This will provide greater analytical sensitivity during HPLC and LSC analysis and will make it easier to quantify minor metabolites. Typically, small molecules are custom synthesised with a specific activity of approximately 50 mCi/mmol for 14Clabelled and up to approximately 20 Ci/mmol for 3H-labelled compounds.

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+ Requirements

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This will generally depend on the intended applications but for typical in vivo ADME studies in animals 20-30 mCi is normally sufficient. In vitro studies will often only require very small amounts, typically 0.5-1 mCi.

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+ Radiochemical Purity

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Radiolabelled compounds are synthesised and then supplied purified to a particular minimum radiochemical purity (RCP). This refers to the percentage of total radioactivity that the synthesised product (parent compound) represents when compared to all of the radioactivity present in the sample/solution provided. Although absolute (100%) RCP can never really be achieved, it is important to use a compound that is of sufficient RCP for the experiments being undertaken.

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Generally, a RCP ≥ 97% is acceptable for most DMPK-related investigations such as animal ADME and in vitro studies (however, where total radioactivity is being measured as with plasma protein binding for example, an RCP ≥98% is advisable).

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