While traditional drug metabolism stud-ies continue to play a key role inunderstanding the pharmacokinetics,metabolism and ultimate fate of small molecules,new technology has opened the door to study com-pounds directly in man. Use of accelerator massspectrometry (AMS) enables detection of verysmall levels of C-14 allowing its use in humans atvery low doses that pose no radiation risk. This, inpart, has led to new initiatives by the FDA andEMEA encouraging fast track first-in-man studies.Recent FDA guidance on the safety testing ofpotentially toxic metabolites has provided anothergrowth driver for C-14.

Regulatory authorities require that studies fol-low official guidance and meet statutory qualitystandards such as GLP, GCP and GMP. Whenusing a C-14 compound it is vitally important toensure there are no impurities present that maygive the appearance of persistent drug residues,lead to artefacts or spurious results. Such adversedata may impact on a drug’s future marketabilityand could lead to costly additional studies.

With increasing demand for quality C-14 com-pounds suitable for human studies, several of theleading custom labelling suppliers have invested inadditional capacity, facilities, processes and analyt-ical capability.

Radiolabelled compounds are considerably

more expensive than their non-labelled analoguesfor the reasons set out below. Taking short cuts inthe radiolabelled synthesis may result in savings,however, these are often insignificant compared tothe cost of a study and may compromise the ulti-mate value of the information and data generated.

Carbon-14 for quantificationCarbon-14 remains the isotope of choice as atracer being biologically equivalent to carbon-12,having a low background in the biosphere (1ppt:1 atom in 1012) and importantly facilitating easyquantification. Its soft �-emission does notrequire shielding but is easily detectable allowingmass balance determination of the parent com-pound and importantly its metabolites (even ofunknown structure) and gives good definition forautoradiography. Its half-life of 5,760 yearsmeans it is unnecessary to correct for decay dur-ing the experiment and provides good countingefficiency using liquid scintillation counting(LSC). Other advantages include minimal chanceof isotope exchange and minimal isotope effectson metabolic transformations.

The new accelerator mass spectrometer (AMS)technology relies upon the ratio of C-14/C-12 forquantification. AMS enables detection of verylow levels of C-14, several orders of magnitudebelow that of LSC, requiring a typical dose of

By Dave Roberts

Drug Discovery World Winter 2009/10 53

Radiolabelling

CUSTOM CARBON-14RADIOLABELLINGinvesting to meet new challenges

One consequence of an increasing demand from regulators for robustquantitative data on the behaviour of new drugs in man and for information onthe fate of pharmaceuticals in the environment, has been an increase in demandfor carbon-14 labelled compounds. This article focuses on the new uses for C-14 compounds, factors impacting on their preparation and how C-14 customlabelling suppliers are responding to meet new demands.

270nCi (10kBq) per individual. With such lowlevels it is very important to avoid any risk of C-14 contaminants. With this in mind, test materi-als are best prepared at high specific activity(~50mCi/mmol; 1.85GBq/mmol) with full analy-sis including structure confirmation prior to dilu-tion with unlabelled material.

A few years ago all the focus for AMS was onphase zero microdosing with drug levels at 1/100th

of the therapeutic level (cold drug) with a micro-tracer level of C-14 to provide a tool for quantifi-cation. Advocates of the technology now focus onpromoting the use of AMS to address specificproblems where the added value is clear. For exam-ple, as an add-on to a traditional Phase I studywhere a microtracer of C-14 is used with a thera-peutic dose. This can provide information on themetabolic profile in man at a very early stage andconfirm that the pre-clinical data from otherspecies is representative.

Recent literature has described use of sensitivelaser-based isotope analysis to count [C-14]CO2. However, it remains to be seen if thisoffers an alternative to AMS for analysis of bio-logical samples.

As part of the IND package there is a require-ment to perform a traditional human mass balancestudy. This will usually be performed on sixhealthy male volunteers using ~100µCi (3.7MBq),per individual, of C-14 radiotracer.

Pre-clinical studies using C-14 are performed bymany of the leading contract research companiessuch as Charles River, Covance, XenoBiotic

Laboratories and Harlan, using C-14 compoundssupplied by external suppliers or internal isotopegroups. AMS analysis is performed, for example,by Xceleron and Vitalea.

Pharmaceuticals in the environmentGiven that many pharmaceuticals need to berobust enough to pass through the stomach it isnot surprising that they find their way into theenvironment via excretion and the public sewagesystem. This has led to increased attention on theenvironmental fate of pharmaceuticals (and otherchemicals) from regulators to identify and mitigatepossible adverse environmental effects prior tocommercialisation. Most major pharmaceuticalcompanies now model the environmental behav-iour of their development compounds. It has longbeen a requirement to provide environmental fatedata as part of the regulatory process for pesti-cides. The experimental protocols used to generateenvironmental fate information such as biodegra-dation/accumulation are robust and well-validatedand are set out in regulatory guidance documents,for example OECD 308. In this guidance C-14radiolabelled compounds are the preferred tools tostudy the degradation pathway and establish amass balance. More recent guidance specificallycovers chemicals found in the aqueous and sludgecomponents of sewage (OECD 314).

The EMEA issued a guideline on the environ-mental risk assessment of medicinal products in2006. It sets out a tiered approach: Phase I requiresan estimate to be made of the environmental risk inthe aquatic component using a predicted environ-mental concentration (PEC) formula. If this is trig-gered then Phase IIA requires basic environmentalfate data in which C-14 radiolabelled compoundsare used in studies, which should be performed toGood Laboratory Practice (GLP) in line withOECD 308. These then may trigger a Phase IIBdata requirement.

Specialist CROs such as BrixhamEnvironmental and Battelle UK Ltd provide guid-ance and perform regulatory studies using carbon-14 on pharmaceuticals.

Basic considerations on radiolabelling with C-14Direct irradiation of suitable nitrogen containingprecursors was ruled out as a possible short-cut tocarbon-14 compounds following extensiveresearch, which found that the C-14 atom wasinserted randomly, in low yield, with formation ofextensive impurities. Thus the only practicalmethod for introduction of a C-14 label is by total

Figure 1

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synthesis of the desired radiolabelled compoundstarting from a single C-14 atom precursor.

C-14 is a reactor-produced isotope requiringlengthy exposure of a nitrogen precursor to highflux neutron beam (14N(n,p)14C reaction) fol-lowed by isolation and purification to barium [C-14] carbonate for distribution.

Given valuable C-14 starting material and thehigh cost of licensed radioactive waste disposal, itis imperative to carry out a practical trial run of theentire synthetic route with unlabelled reagents (theoverall radiochemical yield can be reduced dramat-ically even if a small quantity of impurities arepresent) prior to performing the radiosynthesis.This demonstrates that the synthesis is practicaland minimises radioactive waste production. Therequirement for high specific activity often pre-cludes dilution with unlabelled material and thusthe radiochemist is typically working to prepare~100mg of final product.

Each labelling project is unique and while ideal-ly the radiolabel would be introduced in the lastsynthetic step, in practice, as the label position isoften required in the core of the molecule, the labeloften has to be introduced early in the syntheticsequence. The challenge for the radiochemist thusbecomes one of synthetic chemistry: to devise themost efficient route in terms of radiochemical yieldand number of synthetic steps from barium car-bonate. Radiochemists start much further back inthe synthesis than traditional medicinal chemists asbasic building blocks must be freshly prepared andin any event must derive from C-14 barium car-bonate. For example, synthetic reasons often dic-tate use of simple single carbon precursors such aspotassium [C-14] cyanide as starting material.

The radiochemist may employ process typechemistry, for example, when preparing aromaticmoieties. Consider the sequence to a fluo-robenezene starting with barium carbonate ⇒ bar-ium carbide ⇒ acetylene ⇒ benzene ⇒nitrobenezene ⇒ aniline ⇒ fluorobenzene: sixsteps each involving handling volatile radioactivematerials. Further complexity occurs when consid-ering the additional issues of isomerism arisingfrom the position of the C-14 atom. In the caseabove the aromatic moiety may have moleculeswith differing numbers of C-14 atoms, dependingupon at which stage the carbon-14 is diluted withunlabelled material. As a result this uniformlylabelled material will exhibit multiple mass ions inthe mass spectrometer. Detection sensitivity mayrequire a single mass ion and in this case an alter-native route must be followed to produce singlylabelled material.

The synthetic route provides control over thespecific labelling position allowing study of themetabolic fate of different parts of a molecule, pro-viding confidence in the fate of the parent andfacilitating metabolite identification.

C–14 compounds may needrepurificationCompounds labelled with C-14 have a liability todecompose under the influence of their own radia-tion; termed radiolysis, this effect is independent ofchemical stability. The primary effect when the dis-integrating radioactive atom transmutates resultsin total disintegration of a single molecule and mayproduce reactive fragments. The secondary effect isfrom the �-radiation that may produce ions andradicals, which can react with other molecules orinteract with solvent if present. Knowledge of radi-olysis is empirical and cannot be predicted withconfidence prior to synthesis of the compound.However, the effect of radiolysis can be mitigated,but not eliminated, by storage at low temperatureunder an inert gas. Storage in a solvent in solution,such as ethanol, may help. Thus, there may be aneed for regular purity checks to ascertain if re-purification is required prior to the study. Puritychecks are especially important if studies areplanned over several months to ensure the materi-al is within specification prior to each study.Discolouration is not necessarily an indication ofdecomposition as free radicals may get trapped inthe crystal lattice leading to marked colourationwith no impurities detectable. Figure 2

Drug Discovery World Winter 2009/10 55

Radiolabelling

Radiolabelled API: GMP conceptsThe objective of human ADME studies is todetermine the mass balance of a drug and to eval-uate its metabolism. Such studies require admin-istration of a radiolabelled compound and aresubject to a number of regulations summarised inFigure 1. The exact applicability depends on thenature of the study, for example administration ofmicrotracer quantities do not require ARSACethics approval.

This process for preparation of radiolabelledmaterial suitable for administration to humans isshown in Figure 2.

The radiolabelled component is usually preparedonly once, on a very small scale (less than onegramme) and in a single batch. It is also subject tosignificant dilution with unlabelled material. Thepreparation of the radiolabelled component, theradiolabelled API, is thus not subject to full GMPmanufacture but to Section 19 of the GMP guid-ance which states that the controls should be con-sistent with the stage of development of the drugproduct and that appropriate GMP conceptsshould be applied.

The relevant guidance has been issued in Europeas Eudralex volume 4 and recently in the USA bythe FDA under their Guidance for Industry CGMPfor Phase 1 Investigational Drugs.

This guidance places the emphasis on avoid-ing cross contamination, on accurate recordkeeping and ensuring that the material is wellcharacterised. Process validation is not appro-priate for a single batch so less emphasis isplaced reproducibility or consistency and envi-ronmental controls.

Preparation of the radiolabelled API mayinvolve a new synthesis or repurification ofDMPK radiolabelled material (Figure 3) underGMP concepts. It is preferable to carry out thesynthesis at high specific activity to provide cer-tainty on the position of the C-14 atom and toensure knowledge of C-14 impurities thereby min-imising any problems that might arise during theanalysis of the clinical samples.

Once the radiolabelled API has been preparedand fully characterised, the clinical laboratory per-forming the study accepts the material and pre-pares the final dosage form using a GMP-licensedmanufacturing process under the control of aQualified Person (QP) immediately prior to admin-istration. It is good practice to test the final prepa-ration using LSC and to keep a parallel sample asa check.

If a radiolabelled intravenous dosage form isrequired (ie for absolute bioavailability studies)then an additional step is needed. As terminal ster-ilisation, using irradiation or heat treatment is notsuitable for sensitive C-14 compounds, a GMP val-idated aseptic process involving filtration isemployed to prepare the sterile dosage form (theinvestigational medicinal product (IMP)).Examples of companies which are licensed by theMHRA to perform this in the UK are SCM Pharmaand HMR.

Successful C-14 GMP concepts projects rely upona smooth exchange of documentation between thesponsor, the clinical unit performing the clinicalstudy and the radiolabelled API contractor.

Figure 3

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Figure 4

Custom C-14 radiolabelling companiesThe suppliers of custom C-14 radiolabelled com-pounds have responded to the market for increasedquality and demand for GMP concepts radiola-belled APIs. The following section provides someselected examples of how key suppliers haveinvested to meet this demand:

Selcia has grown dramatically since it began life asan internal isotope laboratory within May &Baker (Rhone-Poulenc). This radiolabelling groupwas part of a spin out from Aventis in 2001 and,following a management buyout in late 2005,Selcia has invested heavily in expanding its customcarbon-14 radiolabelling business with the resultthat it has grown to be one of the leading inde-pendent custom-14 radiolabelling groups. Itrecently announced the opening of a newradiosynthesis laboratory (Figure 4) in 2009bringing its capacity to 29 radiochemists. Selciaspecialises in preparing radiolabelled API in accor-dance with GMP concepts and supplies materialto clinical Phase I units in Europe and the USwhich perform the final IMP production and QPrelease just prior to human administration. Selcia’sanalytical laboratory is GLP accredited by theMHRA and offers GLP NMR and GLP certifica-tion of its C-14 products and GLP certification ofunlabelled material. One advantage of being anindependent C-14 supplier is that clients are ableto choose from the widest number of CROs andclinical Phase I units the most appropriate special-ist for their study.

Quotient provided the main news in 2009 whenit announced it would be acquiring the formerAmersham custom carbon-14 radiolabellingbusiness from GE Healthcare. Amersham hadbeen the leading supplier of custom carbon-14compounds with a long history going backmore than 50 years. Quotient announced itwould be building a new facility to house thisbusiness at a cost of £15 million within 10km ofthe old Cardiff facility. Quotient also plans toretain its existing radiolabelling group (previ-ously known as Biodynamics) inNorthamptonshire. Biodynamics was one of thefirst custom radiolabelling companies to belicensed to manufacture radiolabelled IMPs andhas its own QP to perform release to the clini-cal lab. Quotient is now focusing on offeringclients an integrated service. As well as radiola-belling it can perform Phase I clinical tests andperform non-AMS C-14 bioanalysis, albeit notat a single location.

Drug Discovery World Winter 2009/10

Radiolabelling

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Another radiolabelling group to be acquired by aCRO is the former Eagle Picher group (ex ChemSyn) which is now part of Aptuit. Following theacquisition it has moved to new laboratories inKansas City, Missouri.

Almac has more than 2,500 employees and addeda radiolabelling group, based in Craigavon UK, toits CMC supply chain activity in 2005. LikeQuotient it favours the integrated model and hasrecently announced a broader early stage drugdevelopment package by partnering with a CRO,Covance. In 2009 Almac announced it wasexpanding its radiolabelling team with three newlaboratories and publicised that it has an MHRAGMP licence for radiolabelled IMP (Drug Product)manufacture and formulation including QP releaseto the clinical trials facility.

PerkinElmer acquired the former NEN (NewEngland Nuclear) radiolabelling group based inBoston, Massachusetts (once part of DuPont) in1996. In 2008 PerkinElmer announced it wasexpanding its radiochemical manufacturing facilityand relaunched custom radiolabelling under the‘Under One Roof’ label. It has been very activepublicising its ‘elite’ custom synthesis service for

radiolabelled API to GMP in compliance with FDAPhase I guidance and has opened a second GMPfacility to meet demand. Unlike the other supplierscovered here, PerkinElmer also offers C-14 cata-logue items under its NEN-Radiochemicals brand.

These companies and a number of other suppli-ers are active participants of the InternationalIsotope Society which promotes radiolabelling anduse of isotopically labelled compounds.

Clinical labs using C-14The nature of this work lends itself to specialistcompanies which are expert in their field andlicensed to work with C-14. The radiolabelled APIcan be sent to the most appropriate specialist toperform the Phase I clinical trial.

An example of a Phase I unit licensed to use C-14 is LCG Bioscience, other Phase I unitslicensed to perform the GMP manufacture and for-mulation of the radiolabelled IMP include:Hammersmith Medicines Research (HMR),housed in a new 100 bed clinic; PRA Internationalbased in Netherlands; SGS in Belgium andCovance and HLS in the UK.

Analysis of C-14 clinical samples can be per-formed, for example, by Charles River in Edinburgh.

Internal radiolabelling groupsMany of the large pharmaceutical companies haveone or more internal radiolabelling groups (Table1). They can benefit from outsourced overflowbuffer capacity, which can be available immediate-ly to meet urgent requests and ensure projects arenot delayed by a lack of resources. When largepharma acquire a project from a biotech, their firstrequest may be for GMP radiolabelled API andwith no internal experience of the project, aswould normally be the case for an internal project,an outsourcing company provides a very cost-effective solution. It is no longer unusual for inter-nal groups to consider outsourcing GMP conceptsradiolabelled API synthesis.

Many mid-size pharmaceutical companies regu-larly outsource their custom radiolabelling, some-times using FTE arrangements to ensure resourcesare immediately available to work on their proj-ects. One advantage of carrying out route develop-ment in advance is that radiolabelling can beremoved from the critical development path.

The number of internal isotope groups has beenin steady decline for decades. A consequence ofindustry consolidation has lead to closure of iso-tope groups while formation of new groups hasbeen hampered due to entry barriers such as therequirement for licences, decommissioning liability

ReferencesABPI: Guidelines for Phase 1Clinical Trials 2007.http://www.abpi.org.uk.EMEA: Guideline on therequirements to the chemicaland pharmaceutical qualitydocumentation concerninginvestigational medicinalproducts in clinical trialsCHMP/QWP/185401/2004final.EMEA: Guideline for theEnvironmental RiskAssessment of MedicinalProducts for Human Use. (1June 2006).FDA: Guidance for IndustryCGMP for Phase 1Investigational Drugs (July2008).FDA: Guidance for IndustrySafety Testing of DrugMetabolites (February 2008)http://www.fda.gov.

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Abbott

Astra Zeneca

Bayer Schering Pharma

Boehringer Ingleheim

Bristol-Meyers Squibb

Eli Lilly

GlaxoSmithKline

Johnson & Johnson

Merck & Co Inc

Merck Serono

Novartis

Pfizer

Roche

Sanofi-aventis

Table 1: Large Pharma with internal isotope groups

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and not least difficulty in acquiring trained radio-chemists. Examples of internal radiolabellinggroups closed over recent decades are shown inTable 2.

A complicating factor is that isotope laborato-ries do not always fit comfortably in an organisa-tion as they cover research, pre-clinical and devel-opment functions, often with conflicting priorities.

Also, isotope groups cannot be moved easily aslaboratories have specific licences and if centralisedcause added problems on divestiture.

ConclusionSo what of the future? Consolidation continuesapace in big pharma with the mega-mergers ofMerck-Schering Plough-Organon and Pfizer-Wyeth. Eli Lilly and GlaxoSmithKline haveannounced outsourcing initiatives and Sanofi-Aventis a reorganisation of its R&D. All haveinternal isotope groups so we wait to see whatimpact, if any, this has on their custom radiola-belling activities.

What is certain is that the custom radiolabellingcompanies have taken the view that outsourcing willcontinue to grow and have invested to ensure theyremain ahead of the curve and are ready to meet theincreased demands of their clients. DDW

Dave Roberts is Business Development Directorwith Selcia Ltd. Starting as a synthetic chemist hespent more than 20 years with Rhone-Poulenc andAventis. In 2001 he was a founder of the spin-outwhich was to become Selcia in 2005 and has beenclosely involved in the expansion of their radiola-belling business.

COUNTRY LOCATION ONE-TIME OWNER

Fr Romainville Roussel Uclaf

ITA Verono Glaxo Spa

UK Beckenham Burroughs Wellcome

UK Billingham Zeneca Specialities

UK Brockham Park Beecham

UK Dagenham Rhone-Poulenc

UK Great Burgh Beecham

UK Greenford Glaxo

UK Harlow Smith Kline Beecham

UK Horsham Ciba Geigy

UK Milton Keynes Hoechst

UK Nottingham Boots

UK Ware Glaxo

UK Welwyn Roche

UK Welwyn Smith Kline French

USA Ann Arbor, MI Parke-Davis, Warner Lambert

USA Cincinnati, OH Hoechst Marion Roussel

USA Chesterfield, MO Pharmacia

USA Kalamazoo, MI Pharmacia Upjohn

USA King of Prussia, PA Smith Kline French

USA Palo Alto, CA Roche Bioscience (Syntex)

USA RTP NC Burroughs Wellcome

USA Skokie, IL Searle

USA Summit, NJ Ciba Geigy

Table 2: Pharma isotope groups closed since 1980