In the ever-changing, technology-driven pharmaceutical industry, carbon-14 labeling remains “hot” and is still the first-choice technology for labeling active pharmaceutical ingredients (APIs) and their metabolites. Labeling is a critical technology for APIs and allows quantitative information to be gathered on adsorption, distribution, metabolism and excretion (ADME), which is vital for progression through clinical studies.

However, modern APIs and their delivery methods are becoming increasingly more complex resulting in synthetic challenges for making these radiolabeled products. The technological demands on the isotope laboratory are intensified and new technologies are being used more routinely.

The trend for NCE API innovators to form outsourcing partnerships with CMOs offering isotope labeling services has increased significantly over the last decade, particularly where companies offer integrated technology solutions.

Carbon-14
The choice of isotopic labels for APIs varies, but typically includes carbon-14, tritium, deuterium, carbon-13 or nitrogen-15. This article will focus primarily on carbon-14 studies. Carbon-14 was first discovered by Martin Kamen at Berkeley, by the bombardment of carbon-13 with deuterons, on February 27, 1940. With its long half-life (>5000 years), its low energy but easily detected beta emission, and its chemical nature present in the backbone of all organic molecules, it has become the mainstay of radiolabeling pharmaceutical drugs since the first tracer study by Melvin Calvin, also at Berkeley. He was later awarded the Nobel Prize in 1961 for discovery of the properties of carbon-14.

This radiotracer allowed Calvin to elucidate how plants use carbon dioxide in the process of photosynthesis, a process now known as the ‘Calvin Cycle’. The fundamental importance of these discoveries was recognized in 1946 with the foundation of the first nuclear reactor at Oak Ridge National Laboratory enabling commercial production of carbon-14 for clinical applications.

Today, carbon-14 is produced in a nuclear reactor by the continuous bombardment of an aluminum nitride (AlN) target with a flux of thermal neutrons. This transformation takes at least two years and after processing provides 80-95% carbon-14 enriched barium carbonate (Ba14CO3) with a specific activity of 50-60 millicuries per millimole.

Figure 1 summarizes some of the fundamental chemical transformations used to prepare radiolabeled building blocks such as acetylene, cyanide, cyanamide and carbon dioxide, which are utilized in the synthesis of labeled versions of APIs.

“Hot” radiolabeling of small molecule API
During the last two decades, the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA), have approved a total of 595 new entities, on average 25 new chemical entities (NCEs) and five biologics each year. In 2016, 15 NCEs and seven biologics were approved by the FDA alone. Each of these NCEs will have had a hot labeled version manufactured in house or out sourced to a labeling partner.

Radiolabeled APIs manufactured under non-GMP conditions may be used in pre-clinical studies to evaluate the ADMET profile. In vivo human AME studies require production of the radiolabeled API and investigational medicinal product (IMP) to cGMP standards.

These mass balance excretion studies in humans are a standard part of the development process for new drugs. From these studies, the fate of drug-related material is obtained: mass balance, routes of excretion, and with additional analyses – metabolic pathways.

Carbon-14 labeling plays a vital role in supporting the development of these NCE APIs and their ADMET (absorption, distribution, metabolism, excretion and toxicity) studies due to:

• Easy detection at low levels by scintillation counting. This is becoming of critical importance for studies where doses close to the pharmacological threshold are commonly seen;
• Stability advantages compared with tritium due to replacement of atoms in the molecular backbone rather than the exterior protons which are more susceptible to exchange; and
• Lower specific activities, which reduces destructive side reactions—autoradiolysis.

For an unlabeled synthesis, the chemist has the luxury of a vast selection of cheap and readily available reagents from a commercial supplier, which may be used in stoichiometric excesses, where necessary. Conversely, the labeled synthesis is limited to the fruits of the barium carbonate tree, as highlighted in Figure 1.

Investigational medicinal product (IMP)
In addition to labeling the API, the hot API also needs to be formulated into drug product. For example, in human studies the complexity of the formulation will depend on the needs of the mass balance study. Simple formulations can involve hand weighing of API powder into bottles or capsules. However, more complex formulations may be required to ensure that the radiolabeled IMP is representative of the unlabeled version, e.g. milling, blending with excipients and encapsulation, amongst others. The physical form and particle size of the radiolabeled API may also be critical considerations. To provide confidence on these issues it is desirable to directly analyze the radiolabeled API using physical methods such as x-ray powder diffraction (XRPD) and laser diffraction, specialist techniques not available to many laboratories and CMOs. 

Micro-dosing
Carbon-14 labeling has recently found a new application as a result of advances in detection technology. Accelerated mass spectrometry (AMS) studies, although currently expensive are extremely sensitive and involve a sub-therapeutic ‘microdose’ of radiolabeled API. This technique analyzes the ratio of carbon-14 to carbon-12 to obtain data on pharmacokinetics and metabolism. The extremely high sensitivity of AMS allows human micro-dosing to be carried out using much lower levels of the API and also therefore of radioactivity. This means significantly decreased exposure and waste issues, as well as reduced material requirements. It may also be feasible to use non-GMP materials in these studies.
The manufacture of carbon-14 labeled API for first-in-human AMS studies requires small scale synthesis and handling. For example, a recent project involved the synthesis of 2 mg of a carbon-14 labelled peptide consisting of 84 amino acid residues and representing 1.5 MBq activity—an ample amount for an AMS micro-dosing study, which typically requires in the order of 10 kBq. In comparison, human mass balance studies require doses in the order of 4 MBq per individual.

Tagging biomolecules
Introduction of carbon-14 into biomolecules may involve preparing products with the radiolabel in the linker and/or drug payload moieties. This approach is used in radiolabeling of antibody drug conjugates (ADCs) containing a cytotoxic drug payload. As the range of biological targets increases, so the demand for synthesis of radiolabeled versions expands to include carbon-14 peptides, modified carbohydrates, polysaccharides and glycoconjugates, in addition to PEGylated materials, BIOTINylation and biopolymers. All these technologies are considered specialist areas in their own right, and can be very challenging and require close collaboration between the subject matter specialists and the radiochemists.

Peptides
The design of a radiolabeled peptide manufacture will aim to introduce the labeled residue(s) as late as possible in the synthesis, so that most of the manufacture may be performed by a conventional peptide chemist using established procedures. The simplest way to then introduce carbon-14 into the peptide is via a glycine residue. Transfer of this technology to the isotope laboratory involves optimization to reduce the typical excess of amino acid. Carbon-14 labeled glycine is readily prepared in a high yielding process. Glycine can be labeled at either or both carbon atoms to yield specific activities of up to 120 millicuries per millimole. To achieve the required specific activity for large peptide molecules, it may even be necessary to label more than one residue, or to label a different residue to increase the number of label sites available.

Other more complex peptides with secondary and tertiary structure will require further chemistries after the label incorporation to facilitate this. For example, the formation of disulfide bridges within the peptide will create ‘protein folding’ containing the carbon-14 label. A recent example contained a carbon-14 labeled amino acid incorporated into a 16mer peptide. The cysteine residues within the peptide were interconnected by the selective formation of two disulfide bridges to generate a fold (Figure 2).

Conclusion
Carbon-14 remains “hot” in the industry and the go-to technology for obtaining quantitative data on absorption, distribution, metabolism, excretion and toxicology for APIs. It has become advantageous for NCE API innovators to form outsourcing partnerships with CMOs offering isotopic labeling services alongside specific chemical expertise, specialized technology, and quality systems to ensure that all aspects of the manufacture are fully addressed. These one-stop CMOs can complete a range of tasks to assist the progression of the labeled API from pre-clinical through clinical phases. 

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Dr. Sean L. Kitson is an Investigator of Radiochemistry at Almac and has more than 15 years’ experience in the synthesis of carbon-14 radiolabelled compounds. He is also the 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 Labelled Compounds and Radiopharmaceuticals Award for radiochemistry. sean.kitson@almacgroup.com

Dr. David Speed is a Team Leader of Radiochemistry at Almac and has more than 10 years’ experience in the GMP synthesis and analysis of carbon-14 radiolabeled compounds. He also brings IMP manufacturing experience to the group and is one of the on-site Radiation Protection Supervisors. david.speed@almacgroup.com

Prof. Tom Moody is the Vice President of Technology Development and Commercialization at Almac and Arran Chemicals. He is responsible for driving new technology processes from conception to commercial scale-up across multi-disciplinary research including biocatalysis, flow chemistry, radiochemistry, custom synthesis and commercial production. His work has earned him numerous accolades and he is co-author and author of >60 publications and patents.  He is a strategic leader and technical expert in chiral chemistry and biocatalysis with more than 18 years of extensive academic and industry experience. tom.moody@almacgroup.com

Dr. William Watters is Isotope Chemistry Manager at Almac and has over 20 years’ experience in the Pharmaceutical Industry spanning disciplines as diverse as carbon-14 radiochemistry/stable labelled synthesis, medicinal chemistry, process development, impurity identification/synthesis design, from mg through to multi-kg scale. william.watters@almacgroup.com