Synthesis of Radiolabelled Compounds for Clinical Studies
Regulatory requirements, quality-related measures as well as key manufacture, control, and release aspects for the synthesis of radiolabelled drugs for administration to human volunteers as part of clinical human ADME studies are discussed in detail. Additionally this review provides a general overview of synthetic, technical and methodological aspects to be considered for the synthesis of Tritium- and 14C-labelled compounds. Chemical and biochemical methods and new trends for isotope labelling are discussed based on published examples.
Short-lived radionuclides like 11C and 18F are used for PET (positron-emission tomography) (Cherry 2001) to study mostly in noninvasive experiments the drug passage over the blood-brain barrier, selective accumulation in critical organs, receptor occupancy, dose response or tumor metabolism, and proliferation rates (Rösch 2003). However, labelling syntheses using these short-lived isotopes require specific considerations which are not the subject of this chapter.
Microdosing studies are commonly performed to collect pharmacokinetic data for drug substances following their in vivo administration at sub-pharmacological (trace) doses, a dose at which no pharmacological effect is predicted (Bae and Shon 2011). One method that has evolved significantly over the last few years for determining these data uses a labelled 14C-tracer and a very sensitive detection instrument, the accelerated mass spectroscopy (AMS) technology (Vogel 2000; Vogel et al. 2007). AMS permits the option of decreasing the radioactive dose by a factor of 1,000 from around 50 μCi to about 50 nCi (Garner 2000). The very high sensitivity of AMS permits the evaluation of microdosing approaches including subtherapeutic doses (Lappin and Garner 2003). Smaller clinical doses and/or fewer radioactive administrations to humans may also change the regulatory view on the synthesis of 14C-labelled compounds. The current downside of AMS is that all samples need to be converted into solid graphite, which is an expensive process and only a few laboratories are offering this service. Consequently AMS is not yet used as a standard analytical technique. At the moment, pharmaceutical companies conducting ADME studies apply AMS only when it appears to be absolutely necessary, e.g., in case of high potency drugs, large natural products, or compounds with a very long biological half-life (Lappin et al. 2006). An additional disadvantage is that the same human samples can’t be used for AMS quantification and metabolite identification.
In a recent investigation, researchers determined the absolute oral bioavailability of AZD5122 (Hickey et al. 2016). To assess these values, an intravenous (IV) and oral administration study is mandatory. In the past, absolute bioavailability studies have been conducted by both arms, and for insoluble drugs especially tremendously, efforts have been made to develop a suitable IV formulation, which itself needed further toxicological testing. By applying AMS technology into this microdosing study, the therapeutic oral dose was coadministered with a radioactive IV tracer. Due to the low dose, the IV formulation development was much simpler. By this method, very small differences between 12C and 14C can be quantified; however, the AMS analysis is still mainly outsourced to specialized CROs due to the high financial investment necessary. Nowadays, AMS technology is also used to study biomolecules such as insulins in human ADME studies (Salehpour et al. 2010).
In light of the recent FDA guidance “safety testing of drug metabolites,” stable isotopically labelled (13C, 15N, 2H) analogues can be used more frequently to obtain quantitative and qualitative information on drug metabolism in early human ADME studies (e.g., First in Man) even without specific studies administering radiolabelled drugs (Mutlib 2008). Modern LC/MS technologies and hyphenation of liquid chromatography with CRIMS (chemical reaction interface mass spectrometry) (Jorabchi et al. 2005) presents an opportunity to perform quantitative measurement of metabolites even in the absence of authentic standards or radiolabelled compounds (Abramson et al. 1996).
In these tracer studies, the stable isotopically labelled IV dose differs by mass from the parent drug. The plasma samples from these tracer studies are analyzed by standard HPLC-MS/MS instruments and an isotopically labelled internal standard. By this method, the levels of the labelled IV and of the orally administered drug are determined, and the absolute oral bioavailability can be calculated (Cannady et al. 2016).
General Aspects to Be Considered for the Synthesis of 14C-Labelled Compounds
Technical Considerations for 14C-Labelling
Carbon-14 has many of the properties of an ideal tracer nuclide for human ADME studies (Catch 1961). Because of its very long half-life (5,730 years), it is unnecessary to correct for decay, and 14C-labelled compounds can be prepared and stored for a long period if radiolytical decomposition can be minimized (see also under “Synthetic Considerations for 14C-Labelling”). Carbon-14 decays to nitrogen-14 with the emission of a β-particle (maximum energy, 156 eV; average energy, 49 eV). This emission is sufficiently energetic to make measurement at moderate specific activity fairly simple but weak enough to make shielding unnecessary. The range of these soft β-particles is about 15–16 cm in air and 0–2 mm in a solid medium. This means even at very high specific activities, 14C-labelled compounds can be safely handled in standard glass vessels and conventional lab equipment if reasonable radiation safety precautions are taken. However, in most countries, working with radioactive materials requires strict reporting, licenses, and/or authorizations from authorities. Local regulations may be different or stricter with respect to the handling of radioactives, to containment equipment and to lab facilities.
Synthetic Considerations for 14C-Labelling
In general, any organic compound that can be synthesized can be labelled, but specific labelling of more complicated molecules may be difficult and expensive. Usually, a target directed total synthesis approach is required for 14C-labelling of drug development candidates. For selecting the right labelling position, several aspects have to be carefully considered. The position of the label should be away from sites which are chemically unstable and away from sites of metabolic attack in order to ensure the label is kept in the main metabolic fragment. In cases where high specific activities are required for the planned study, a double 14C-labelling or alternatively 3H-labelling needs to be considered because the maximum achievable specific activity for single 14C-labelled organic compounds is 62.4 mCi/mmol (2309 MBq/mmol) (see also Scheme 1 (Schulte 1966)).
As well, the development of a labelling synthesis is dependent on the availability of suitable precursors, the length and complexity of the synthetic pathway, the reliability of the process, as well as radiation safety aspects, e.g., avoidance of volatile reaction components if possible.
Planning of a 14C-Synthesis
Introduce the 14C-label as near as possible to the last step of the synthesis.
Introduce the 14C-label after stereochemical resolution.
Introduce the 14C-label in a known position.
Ensure adequate specific activity and high radiochemical purity of the product.
Provide high radiochemical yields.
Consider synthetic efforts for unlabelled precursor synthesis.
No special laboratory equipment is required, but preparation techniques may differ from conventional synthetic work especially when volatile radioactive precursors are handled or volatile intermediates and/or side products are expected. Further difficulties may increase as the scale of the reactions is reduced, e.g., yield, impurities, solvent content, crystallization.
A 14C-labelled compound for which preparative methods are not well established should be synthesized as follows: First, the pathway is elaborated with non-radioactive material on the desired scale until a reliable process has been developed and the operator is adequately familiarized with the chemistry. Product purity should be checked by the usual chemical and physical methods. Then, the experiment is carried out at the tracer level to establish the nature of impurities and side products (e.g., volatility), to determine the yield of the desired product and to check for radio-accountability. Finally, the procedures developed are duplicated with limited amounts of the 14C-labelled precursor.
Methods for 14C-Syntheses
Many good standard procedures for the chemical preparation of the more common 14C-labelled compounds have been summarized in literature reviews (McCarthy 2000) and in books or in book chapters (Murray and Williams 1958; Heys et al. 2009) (see also Scheme 2). Today, many specialized supply companies provide a variety of these basic 14C-labelled precursors or even more complex molecules on a custom synthesis basis. Usually, the structural complexity of modern drug development candidates requires multistep labelling syntheses. Therefore, a common strategy in the pharmaceutical industry is to purchase or outsource the preparation of basic labelled organic compounds required as starting materials for an in-house synthesis of labelled drug development candidates. If applicable, a late-stage introduction of the 14C-label into an unlabelled advanced intermediate can be highly efficient (see also “Planning of a 14C-Synthesis,” criteria (a)). For example, the Grignard reaction (Knochel 2005) of organo-magnesium halides with 14CO2 or a transition metal catalyzed cyanation (Yu et al. 2017) with metal cyanide and subsequent saponification are convenient methods for introducing labelled carboxyl functionalities (Cao et al. 2007). Other small building blocks frequently used for 14C incorporation into organic molecules are [14C]formaldehyde, [14C]methyl iodide, [14C]thiocyanate, and 14C-labelled acetic acid derivatives (McCarthy 2000; Heys et al. 2009).
After successful introduction of the 14C-label, all further chemical transformations can be performed applying classical or modern organic chemistry approaches as described for unlabelled compounds including asymmetric synthesis (Voges 2002) or cross-coupling reactions (Derdau et al. 2003).
Besides chemical methods, biochemical synthesis offers the ability to obtain labelled natural products and organic compounds not always accessible by conventional synthesis (Evans 1981; Benakis 1994) Oligopeptides, proteins, antibodies, as well as a large number of pharmaceutically relevant compounds, e.g., antibiotics synthesized by fermentation with the aid of yeast, bacteria, or fungi, can be labelled using biochemical methods. Corresponding 14C-labelled compounds can be prepared if the relevant 14C-labelled starting materials, e.g., 14C-labelled amino acids, are employed in the fermentation process. Yields are as important as in purely chemical syntheses, but unfortunately, only a few biochemical processes provide a single 14C-labelled product in high yield (Wallace et al. 1995). Decarboxylation and the production of large quantities of 14CO2 might be another drawback that needs to be considered when planning fermentation for labelling purposes.
The addition of ammonia to [14C]cinnamic acid catalyzed by the enzyme PAL (phenylalanine ammonia lyase) carried out in ammonia buffer at pH 9.8 led to the formation of [14C]L-phenylalanine ([14C]-L-Phe). Subsequently, this intermediate was converted to [14C]PPA employing the enzyme PheDH (phenylalanine dehydrogenase) in the presence of NAD+. As is normal for enzymatic processes, the yield of this reaction strongly depends on the incubation time, concentration of L-Phe, buffer choice, pH, and enzyme quantity. Similarly, conducting the first step with fully deuterated or tritiated ammonia buffer gave [(3S)-2H]-L-Phe and [(3S)-3H]-L-Phe, respectively, which offered the opportunity to get access to the corresponding deuterium and tritium-labelled PPA as well (Skowera and Kanska 2008).
Another biochemical method for preparing labelled compounds is photosynthesis in which 14CO2 is assimilated by, for example, algae such as Chlorella vulgaris (Godward 1960), cyanobacteria such as Anacystis nidulans (Tovey et al. 1974), or plants such as tobacco (Nicotiana sp.) or Canna indica (Putman and Hassid 1952), or by detached or full-sized plants grown in a 14CO2 atmosphere in sealed greenhouses or plastic bags (Benkis et al. 1985). This technique called “isotope farming” can provide high specific activities and good yields of, for example, 14C-labelled glucose, starch, nucleosides, amino acids, and lipids.
Stability of 14C-Labelled Compounds
Compounds labelled with carbon-14 decay to nitrogen-14 with the emission of a β-particle. Since the energy of these ß-rays by far exceeds bond energies of organic molecules, structural damage can occur. If the radiation energy is absorbed by the compound itself, the excited molecule may break up and/or react with other molecules. The activated decomposition fragments may also react in a sort of chain reaction with other molecules producing impurities (Bayly and Weigel 1960; Rochlin 1965). Typical reactions resulting from irradiation are dehydrogenation, oxidation, decarboxylation, deamination, condensation, and polymerization, in many cases through radical reactions (Sheppard 1972). In some cases, the shelf life is reduced from years to weeks or even days.
The rate of decomposition depends on storage conditions, specific activity, and chemical structure. Though it is not yet possible to foresee exactly the behavior of each compound, a thorough stability study may result in storage conditions that provide reasonable shelf life of the compound and thus reduce purification efforts and secure rapid availability of stock material. The following storage rules have proved to be successful and can be applied to reduce decomposition (Evans 1976; Bayly and Evans 1966): optimize storage conditions as regards chemical stability, store at the lowest practical temperature, dilute the specific activity, avoid high amounts of activity, store labelled compounds in solution, add radical scavengers or other stabilizers (Fredenhagen 2002), and avoid unnecessary reopening of vials and warming/cooling cycles.
In spite of all potential precautionary measures, the shelf time of labelled compounds is always limited and requires repurification of the material more or less frequently (Bayly and Evans 1968).
The development of a suitable purification method for 14C-labelled compounds can be crucial because typically a radiochemical purity of >98% is necessary for the planned studies (for specific applications up to 99.8% are required). Additionally, the limited shelf life of 14C-labelled compounds may require repurifying the 14C-labelled compound from time to time. Since synthetic impurities and degradation products can be completely structurally different, it might be necessary in some cases to develop different purification methods as well. Typically, 14C-labelled compounds are purified by column chromatography, semi-prep reversed-phase HPLC or crystallization (Evans 1981).
After purification, the 14C-labelled drug development candidate often needs to be diluted to obtain the specific activity required for the planned studies. To this end, the highly radioactive compound is homogeneously mixed with unlabelled material of the same compound by dissolving both in a suitable solvent. Subsequently, the solvent is removed by evaporation, the product crystallized, precipitated, or lyophilized to afford the diluted 14C-labelled drug development candidate ready for administration or further formulation to the drug product.
After repurification and dilution of the radioactive drug substance, generally an intensive analytical release testing program according to specifications is followed to guarantee consistent product quality (Filer 1988; Dueker et al. 1998). Typically, the identity of the compound is confirmed by NMR (Schenk et al. 2015) and LC-MS; the radiochemical, chemical, and stereochemical purities are checked by analytical HPLC; and the specific activity is measured by LSC and/or high-resolution MS (Schenk et al. 2016). In addition, specific applications or specific compound-related properties may require supplementary analytical tests, e.g., ion chromatography, water content determination, polymorphism, or particle size investigations (Braun et al. 2004).
General Aspects to Be Considered for the Synthesis of Tritium-Labelled Compounds
Synthetic and Technical Considerations for 3H-Labelling
Tritium-labelled compounds are much less frequently applied for human ADME studies (see also “Introduction”) because an essential problem is the integrity of the carbon-tritium bond and the specificity of the labelling. There are however rare examples for clinical trials with tritiated compounds (Straznicky et al. 2014). The high specific activity of 28.8 Ci/mmol of tritium which is approx. 500 times higher than achievable with a single carbon-14 label (62.4 mCi/mmol) is an advantage (Evans 1974). This property makes tritium irreplaceable for labelling of large molecules, particularly biomolecules such as peptides, proteins, oligonucleotides, and antibodies, as well as for early labelling strategies to support discovery purposes.
Tritium labelling is often much simpler than 14C-synthesis and labelling can often be accomplished very late in the overall synthesis. The typical sources for the tritium label are tritium gas or specific tritiated reagents and sometimes tritium water. Modern stainless steel manifolds allow a safe handling and storage of tritium gas (Benakis 1994). However, as for 14C-synthesis, a number of specialized supply companies offer custom tritiation services. In principle, similar points as mentioned under “Methods for 14C-Syntheses” should be considered for the planning of a 3H-synthesis. Compared to 14C-synthesis, the scale of tritiations is even further reduced. Often, only a few milligrams of material is handled, which requires specific preparation techniques, operator training, and elaboration work. The shelf life of 3H-labelled compounds is usually decreased compared to 14C; however, it can be improved by storage in ethanol solution at low temperatures (<−80 °C). However, purification, dilution, and analysis aspects are similar to those already mentioned for 14C-labelled materials (see also “Purification,” “Dilution,” and “Analysis”).
Chemical Methods for 3H-Labelling
Comprehensive literature reviews summarizing synthetic techniques developed for tritium incorporation have been published recently (Heys et al. 2009; Saljoughian and Williams 2000), and therefore, only the main principles will be briefly discussed. There are two basic approaches for introducing tritium into organic molecules: exchange labelling (Atzrodt et al. 2007; Heys 2007; Lockley 2007) and synthetic tritiation methods (Evans 1981; Saljoughian 2002). While there have been several drawbacks applying exchange labelling methods in the past (e.g., lower tritium abundance or the isotope being widely distributed over the molecule), nowadays, newly developed catalysts have proven their unique efficiency for selective tritium introduction (Allen et al. 2010; Nilson and Kerr 2010). As exchange labelling can be highly cost and time efficient when carried out directly on the target molecule, it is a standard method in pharmaceutical laboratories today; however, a suitable catalyst directing group remains essential.
[3H]Vardenafil was synthesized by reduction of a specially synthesized amide precursor with freshly prepared lithium aluminum tritide (Pleiss 2003). For the synthesis of [3H]ragaglitazar at high specific activity, a suitable dihalogenated precursor was synthesized and then the tritium incorporated by a catalytic dehalogenation reaction in the presence of tritium gas (Kristensen et al. 2003). The tritiation step for the synthesis of [3H]Mecillinam was performed by treating the corresponding dehydro-mecillinam, itself prepared via a six-step synthesis, with tritium gas in the presence of 10% Pd/C as the catalyst (Frederiksen and Sörensen 2003). [3H]Ansamitocin P-3 was prepared by alkylation of ansamitocin PDM3 with [3H3]methylnosylate under basic conditions. Ansamitocin P-3 is a precursor of LDM4 which is used to prepare drug-antibody conjugates (Sun et al. 2011). In all cases, specific unlabelled precursor molecules had to be synthesized, and therefore synthetic tritiations are often limited by the chemistry required both prior to and during the labelling process.
Regulatory Requirements for Application of Radiolabelled API to Humans
General Study Requirements
Administering radioactivity to human beings raises general ethical questions due to the well-established carcinogenic and/or teratogenic potential of radioactive compounds (Dain et al. 1994). Radiation exposure should be “as low as reasonably achievable” (ALARA concept), and even when administering small doses of radioactivity, given the residual risk, exposure should be minimized. Therefore, clinical studies involving the administration of radioactive drugs to humans need to be approved by a special ethical committee. Additionally, submission and approval of an Investigational New Drug Application (IND) for the USA or, if the study is carried out in the EU, an Investigational Medicinal Product Dossier (IMPD) is required. These documents include detailed information on the synthesis, medication, and analytical release, as well as a supportive stability study. The regulatory requirements for both the manufacturing process and study design are subject to various local and national authorities and strongly depend on the location and specifics of the study site and the country of the manufacturing facility.
The GMP guide ICH Q7A Good Manufacturing Practice for Active Pharmaceutical Ingredients (API) does not apply to manufacturing/control aspects specific to radiolabelled compounds with long-lived isotopes (e.g., 14C, 3H). However, Chap. 19 contains guidance for the manufacture of APIs used in clinical trials (APIs for investigational use during early phases of development) in general. For EU countries, the EU GMP Guideline Part II Basic Requirements for Active Substances used as Starting Materials including Chap. 19 APIs for Use in Clinical Trials is recommended to be followed although not required by community legislation. During the manufacturing process, the highly radioactive drug substance is diluted with unlabelled API (manufactured according to GMP requirements) to achieve the specific radioactivity required for the planned study. Although typically only very small amounts of radioactive material are incorporated in the final drug substance, it could be considered a radioactive Active Pharmaceutical Ingredient (rAPI). Therefore, in some countries, the manufacture of radiolabelled APIs is covered by national drug laws and ordinances, whereas in others, this has not come within the scope of regulatory GMP inspections and does not require certification. These inconsistent interpretations may result in different levels of GMP (Good Manufacturing Practice) being requested for the synthesis of radiolabelled API’s by different national health authorities.
Consequently, depending on national regulatory requirements, pharmaceutical companies have developed different creative approaches that combine quality principles, radiation safety aspects, and the other challenges inherent in radiosynthesis (Lloyd et al. 2003; Fontana et al. 2000). Approaches including full GMP compliance for the whole synthesis or only parts of it (e.g., only purification and dilution, including in some cases full environmental control), different GMP or GLP-like (Hong et al. 2008), or non-GMP classified processes (Bonacorsi et al. 2007) have been applied. Generally, the local authorities are allowed to inspect the manufacturing facilities to review compliance with the appropriate regulations.
General Quality-Related Measures that Should Be Applied for the Synthesis of Radioactive APIs
Independent of whether GMP is formally required or not, the synthesis of 14C-labelled drug development candidates that are to be administered to humans should follow higher-quality standards than those for in vitro or animal study applications. Thereby, the stringency of quality standards should increase as the process proceeds from the early to final synthetic steps, purification, and dilution. Compared to orally administered compounds, the synthesis of radiolabelled drug substances that will be formulated for parenteral application should be subjected to more stringent control. At least the final steps, such as purification and dilution, or even the last covalent bond modification step, should be performed as a kind of validation to confirm the correctness and quality of the applied methods. Usually all synthetic, manufacturing, analytical, and release activities are reviewed by an independent quality assurance organization to ensure the compliance of all process steps with regulatory and internal company quality requirements. SOPs (standard operation procedures) are established to define the details of all quality-related operations and processes.
Although in principle the labelled material could be any proportion of the administered drug substance (rDS), in practice, it is normally small, typically forming less than 5% of rDS.
Adventitious agents evaluation and a complete review of the synthesis concerning BSE/TSE and viral safety is requested by several authorities. In addition, raw materials, intermediates, solvents and reagents, as well as materials that will come into direct contact with the radiolabelled drug (dry reagents, charcoal etc.) often have to be evaluated by testing or received with a supplier’s Certificate of Analysis (CoA) and subjected to at least identity testing.
The retrievable and traceable recording of all process- and testing-related information (including signatures) in legible documents should be in place. In addition, several affiliations to combined manufacture units require written production instructions and records as well as a full quality assurance review of the synthesis documentation.
All equipment of the radiosynthesis laboratories that are critical to product quality (balances, preparative HPLCs, pH-meters) should be calibrated and serviced at appropriate intervals according to written procedures. Furthermore, the equipment used has to be qualified by a certain formal methodology in order to attest its proper technical status. This is supplemented by the regulations for computerized systems. Hence, for example, the hardware and software of an HPLC has to be evaluated regarding data security, robustness, validated operating procedures, etc.
Glassware and other equipment that will come into direct contact with the radiolabelled compound for clinical use should be new or cleaned according to standardized procedures. Stirrer bars, syringes, needles, and glass pipettes should be new.
The manufacturing facilities including fume hoods and HPLC cabinets should be dedicated and cleaned. Appropriate measures must be taken to prevent product contamination or cross-contamination. In general, a microbiological environmental monitoring of the work surfaces is required. Often due to the contradictory nature of radioprotection and GMP clean room regulations, establishing a GMP clean room facility is challenging (Loewe et al. 2016).
Another significant consideration in the manufacture of API for clinical use is the level of GMP expertise of all staff members. Their knowledge and awareness of GMP compliance needs to be kept to a high standard by continuous training on SOP and guideline compliance, in transparency and accurate documentation, and in hygiene or bio burden. For each employer, specific duties and responsibilities should be recorded in written job descriptions. Finally, a document management system has to be established including deviation and change control reporting, and last but not least, a regular interval of inspections should take place (Filer et al. 2016).
Conclusion and Outlook
Suitable quality measures should be in place in order to ensure that the manufacture, control, and release of radiolabelled compounds administered to human volunteers will satisfy quality requirements and guarantee both patient safety and the reliability of study results. In the last couple of years, the regulatory stringency for the synthesis of radiolabelled compounds for clinical use has increased, and certainly this will continue in the future. Whether radioactive tracers or the AMS technology will be used to study proteins, peptides, and other macromolecules on a frequent basis remains unclear. Up until now, there has been no requirement from the authorities to perform human ADME studies with biologicals in order to gain regulatory approval. However, this translational information might be valuable from a scientific point of view to help support milestones decisions in pharmaceutical drug development.
References and Further Readings
- Allen J, Brasseur DM, De Bruin B, Denoux M, Pérard S, Philippe N, Roy S (2007) The use of biocatalysis in the synthesis of labelled compounds, proceedings of the ninth international symposium on the synthesis and applications of isotopically labelled compounds, Edinburgh, 16–20 July, 2006. J Label Compd Radiopharm 50:342–346CrossRefGoogle Scholar
- Bayly RJ, Evans EA (1968) Storage and stability of compounds labelled with radioisotopes, Amersham Review No 7Google Scholar
- Benakis A (1994) The importance of labelling of bioactive compounds in the development of new drugs. Adv Drug Des Dev:123–136Google Scholar
- Benkis A, Sugnaux FR, Collet FR, Kradolfer GF, Berney JP, Sion C, Necciari J, Cautreels W (1985) Carbon-14 photosynthesis labelling of natural compounds and drugs from plants. In: Synthesis and Applications of Isotopically Labeled Compounds, Proceeding of the Second International Symposium, p 219Google Scholar
- Cannady EA, Aburub A, Ward C, Hinds C, Czeskis B, Ruterbories K, Suico JG, Royalty J, Ortega D, Pack BW, Begum SL, Annes WF, Lin Q, Small DS (2016) Absolute bioavailability of evacetrapib in healthy subjects determined by simultaneous administration of oral evacetrapib and intravenous [13C8]-evacetrapib as a tracer. J Label Compd Radiopharm 59:238–244CrossRefGoogle Scholar
- Catch JR (1961) Carbon-14 compounds. The Radiochemical Centre Amersham/Butterworths, Bucks/LondonGoogle Scholar
- Deroubaix X, Coquette A (2004) The ins and outs of human ADME studies. Bus Brief: Pharmatech:1–4Google Scholar
- Evans EA (1974) Tritium and its compounds: second edition. Wiley, LondonGoogle Scholar
- Evans EA (1976) Guide to the self-decomposition of radiochemicals, Amersham Review No 16, The Radiochemical Centre, Amersham; booklet Amersham International plc. 1992; booklet Amersham Biosciences 2002Google Scholar
- Filer CN (1988) The analysis of 14C and 3H-labelled compounds. Synth Apl Isot Labelled CpdGoogle Scholar
- Fontana E, Dellavedova P, Gambini L (2000) Carbon-14 and tritium labelled compounds good manufacturing practices for studies in humans. In: Pleiss U, Voges R (eds) Proceedings of the seventh IIS symposium Dresden, Synth. Apl. Isot. Labelled Cpd. Wiley, Chichester/New York/Weinheim/Brisbane/Singapore/Toronto, pp 499–502Google Scholar
- Fredenhagen A (2002) Radiochemical stability of 14C-labelled compounds on storage: benefits of Thioethers. J Label Compd Radiopharm 45:211–220Google Scholar
- Heys JR, Voges R, Moenius T (2009) Preparation of compounds labelled with tritium and carbon-14, John Wiley & Sons, Inc. New York.Google Scholar
- Knochel P (2005) Handbook of functionalized organometallics, applications and synthesis. Wiley-VCH, Weinheim.Google Scholar
- Lappin G, Garner RC (2003) Big physics, small, doses; the use of AMS and PET in human microdosing of development drugs. Nat Rev Drug Discov:233–240Google Scholar
- Lloyd C, Potwin J, Wright C (2003) Radiosynthesis conducted under cGMP compliance. In: Dean DC, Filer CF, McCarthy KE (eds) Proceedings of the eight IIS symposium Boston, Synth. Apl. Isot. Labelled Cpd. Wiley, Chichester, pp 251–254Google Scholar
- Loewe C, Atzrodt J, Reschke K (2016) Joe Schofield, conception, realization and qualification of a radioactive clean room lab facility dedicated to the synthesis of radiolabelled API for human ADME studies. J Label Compd Radiopharm 59:611–614Google Scholar
- Murray A, Williams D (1958) Organic syntheses with isotopes. Interscience Publishers, New York. Muccino RR (1983) Organic syntheses with carbon-14. A Wiley-Interscience PublicationGoogle Scholar
- Raaen VF, Ropp GA, Raaen HP (1968) Carbon-14 in series in advanced chemistry. McGraw-Hill Book, New York/St. Louis/San Francisco/Toronto/London/SydnyGoogle Scholar
- Rochat R, Lopez MJ, Tsurugi H, Mashima K (2016) Recent developments in homogeneous organomagnesium catalysis. Chem Cat Chem 8(1):10–20Google Scholar
- Rösch F (2003) Radiochemistry and radiopharmaceutical chemistry in life sciences. In: Vertes A, Nagy S, Klencsar Z (eds) Handbook of nuclear chemistry, Vol 4, Kluwer Academic Publisher, Berlin.Google Scholar
- Saljoughian M (2002) Synthetic tritium labeling: reagents and methodologies. Synthesis 13:1781–1801Google Scholar
- Schenk DJ, Lockley WJS, Elmore CS, Hesk D, Roberts D (2016) Determining the isotopic abundance of a labeled compound by mass spectrometry and how correcting for natural abundance distribution using analogous data from the unlabeled compound leads to a systematic error. J Label Compd Radiopharm 59:136–146CrossRefGoogle Scholar
- Schulte HR (1966) Synthesen 14C-markierter organischer Verbindungen in Radioactive Isotope in der Organischen Chemie und Biochemie. Wiley-VCH, Weinheim.Google Scholar
- Sheppard G (1972) The self-decomposition of radioactively labelled compounds. At Energy Rev 12:3–66Google Scholar
- Straznicky NE, Grima MT, Sari CI, Eikelis N, Lambert GW, Nestel PJ, Karapanagiotidis S, Wong C, Richards K, Marusic P, Dixon JB, Schlaich MP, Lambert EA (2014) A randomized controlled trial of the effects of pioglitazone treatment on sympathetic nervous system activity and cardiovascular function in obese subjects with metabolic syndrome. J Clin Endocrinol Metab 99(9):E1701–E1707CrossRefPubMedGoogle Scholar
- U.S. Department of Health and Human Services Food and Drug Administration Guidance for Industry Q3A Impurities in New Drug Substance (2008) Google Scholar
- Walker AE, Pothuluri JV, Heinze TM, Volmer D, Cerniglia CE (1996) Biosynthetic production of 13C and 14C Erythronolide labeled erythromycin A. J Label Compd Radiopharm 29:59–67Google Scholar
- Wallace MA, Dean DC, Ellsworth RL, Marks T, Mellio DG, Maseurekar P (1995) Studies on the biosynthesis of Avermectins using carbon labelled 2-methyl butyric acids. In: Allen J, Voges R (eds) Synthesis and applications of isotopically labelled compounds 1994. Wiley, Chichester/New York. Paper 108, 605Google Scholar
- Wilson RJ (1966) The radiochemical manual, 2nd edn. The Radiochemical Centre, AmershamGoogle Scholar