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& 14C Radiolabeled Compounds

and their Applications in Metabolism studies

I I nvestigational chemical entities are usually radiolabeled molecules or their
where the labeled atom is usually Tritium (,H,T), Carbon_14
;
('*C). Sulphur-35(35S), Phosphorus-32(32p), Iodine_l25lrrily,Iodine_13r
I II T:ibolttes
(r31!
and kon-59 lsoFe) amongst others. Such isotopicalry laberei
compounds or
I lts
metabolltes act identical to the non-labeled compound
I
and hence such
compoun9: flr9 use as tracers enabling variety of biologicar
i pharrnacokrnetics,
studies such as
I
drug metabolism ADME studies in human and veterinary
j nharmaceuticals. agrochemicals and cosmetics for environmental fate studies,
I wheretn therr degradations are noted.
] Besides these, the labeled compounds are also used to establish biotransfbrmation
I pathways, receptor bindinj, transport processes,
activity and
r multistep biochemicar studies. Increased regulations ine,zyme
the European union
I 6ucu programme: Regulation on Registiation, Evaluation, Authorization
r and Restriction of Chemicars) have resulted in an increased requirement
of
I isotopicaliy labeled molecules.
I rn" isotopes 3H and tac termed soft p-emitters due to their low energy
I radiations. This..has madearethem
Dr. Jagadeesh Setti Guthi
the isotopes of first choice for labering to
GVK Biosciences Private Limited
I understand the distribution, flux metabolic fate and tissue localization of a drug
t, in-viuo, parlicularly preclinical and crinicar studies
in the drug dir"ou"ry
Hyderabad
i development stages. As per US FDA's safety assessment procless, studies und
jogodeesh. uth
Em
gvkbio.
m
can
administration ofradiolabeled drug from l0-l0d pci ,ac
I *l:ly_"lhe
or 50-1000
3H
pci
in healthy volunteers. Greater than g0% of arl existing drugs
II used
have been
in.their labeled form for the above assessments. Arthough,
the issues of
r waste disposal and cost have cast impact, newer less hazardous fluorescent
I and stable isotope labeling techniques have
been developed. Isotope labeling
r continues to be the method of choice.
a iI:

i@

co

Dr. Sharadsrikar Kotturi

GVK Biosciences Private Limited

Hyderabad
E m a i I : Sh o

ra d s ri ko r. kott u ri @ g v k b i o. co m

lHil,:,lHu:

,
I
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rac.labelling of molecures
offers significant advantages compared to 3H rabeled
molecules including, (a) Stability in biological fluids (3H is
ta-olte and possibility

*:li:|. yirt biological water (b) Longer hatf-tife and better counting
?f.
detectabltrty, (c) Lower radio toxicity and (d) From a synthetic
standpoint,
labeling offers greater selectivity, apart from greater ease
of handring and
lia
storage stability.

April201.4

Points to be considered for the synthesis ofradiolabeled

compounds include: selection of isotope considering

the stability in systemic conditions, specific activity,
and the position of labeling keeping the metabolism
in perspective (to track breakdown metabolite or
fragment) all of which considerably impact the cost
and chemical stability of flnal compound.

Physical characteristics

of

(clt)hriC.{
1

MooCS(II2trt'lOMo{-- CHlttCOoH
Hr4coNRrRr

Ittorqi

with specific activity of -S5mCi/mmol. Tritium (3HlTj

is produced in nuclear reactors by neutron activation
of Lithium-6. Both nuclides emit p radiation with a
maximum energy of laC-156 keV and 3H-1g.6 keV,
form stable N (nitrogen) and Helium with half-lives

NHz
I
*r.it'tN
iYHl

Single carbon incorporation (taC),

Various reactions that can

be carried out

from

BaraCO, are shown in the Fig. l.' Natural products

such as sugars are photo-synthetically prepared in the
laboratory by exposing the plant leaves to raCO, in the
presence of water and light, followed by extraction and
isolation of sugars using paper or preparative column
chromatography.2 Synthesis of taC labeled natural
amino acids is possible by growing unicellular green
algae (Scenedesmus acutus)3 and other organismsa in a
medium using 1aC-Glucose and NaHraCO, in solution
as a carbon source.

,,oroH

nl,r'(x{\\ /-*'*uo,'*
\ I,.* .-* Bf.6x{,crrcx,r

-l*o,,*,"*"/t

"

*-\

I
I -,H,,,*o

-\*.,.X,,-O

HEc=,{L:cooR'
.: DGmb$r1c-

M=lhd

T6I'l(NolrtH!

"L,

Blrtllr-r4(xJlBr

] ..

Fig 7: Four'trees' of building blocks derived from BolaCO,

*ffi

R-Gr*crr3

racHrl,NaoEt

flflo'
\-'6

Ci]-o"o'
,s.Nr,il \-'b

K2CO3/ActuDhrIF

R-NH,

pooR

1*t

r
T"Ii',
KtCOs
llcHrl

R-NH-I4H3

lacH"{COOR

l..--.-..---..-".--.*

'cooR

toon I ,*cttI _ ,rcu,vcooR

L*-- / \^^
r+cu/
I

April2Ot4

t\nrs,.m

Hrt(oEr)b ''(}l,NHx

soocr.c=r.ccooH- rt:f,=rt* ..-.* (D

lncorporation of labeled alkyl groups

Methyl iodide ('aCH,I) is regularlyused formethylation,

to prepare methyl ethers of aliphatic alcohols (a) and
aromatic/phenolic ethers in the presence of base. (b)
Methylation of amines with methyl iodide ('aC) carried
out using weak base GqCOr) or a non-nucleophitic
base (c). Further, alkylation of active methylene group
with methyl iodide is possible using bases (NaH, BuLi
amongst others) (d). Additionally, labeled alkyl halides
can be used to generate Grignard reagents followed by
reacting the carbonyl compound (ketones, aldehydes)
to get secondary andtertiary alcohols while CO, yields
carboxylic acids (e).

+rtHJ

11rr,.q<noq1, Mr'cl.lo,itrtit},ls

NHrucs.NHr i\r

R-oH

Labeling Methods

',,*-l=u'*"cfi'Nl
'lcno

I
.--[[4cql

rlq',s'

of 5760+30, 12.30 years respectively. The theoretical

specific activity (inherent property) of Carbon-I4 is
62mCi/mmol and tritium is 29 Cilmmol.

xcltutooH
Rcooc

f
rtl --trq} r/
, \
-

Nlrl-ucr)-Nur

raC, 3H are summarized

below: while the two radioisotopes are naturally

available in traces (ppt, parts per trillion), raC also
produced by high flux neutron bombardment in
nuclear reactors and isolated in the form of BaraCO,

+Hrlcoo' \

tactt
Ketones

'nc(c)n hrer I

Aq!g-

^hoon

lc; 6g

n/".

c-l4trrz4H(RloH
14cur1c1-coom

Fig 2: S.chemotic diogrom of synthesizing vorious

using

laC

1aC

lobeled compounds

lobeled alkyl holides (eg: Methyt iodide:aC)

$pinco Biotech

euttingEdge

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c_

13

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Fig 3: Rodio-TLC ond lmoging sconner (A), Schemotic diogram of chromatogram (B)
compound (s) seporation initial spotting-i, compound-ii and iii

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of

TLC plote

of C, with radiolabeted

I
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Radlffidive

ffi0rund

I
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0 I 2 3 it 5 6 7 I 9 1011121i1{1s1617191910

.. . Rutrtlm {minua$6}

Fig 5: Schemotic diagrom of overlapped

ffi

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S.lflm

wtl!

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Fig 4: lnstruments of diagrom of HpLC and 6-Rom detector

involved in purity determinotion of Rodiolobeled compounds.

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Use of Iabeled precursors

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Several drug candidates and their metabolites can

be synthesized using commercially available radio
labeled precursors viz. aromatic and aliphatic nitro,
amino, cyano and halide derivatives.

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Tritium (3H/f)

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chromotogram
obtoined from online uV ond rodiodctive detectors (P.-RAM)
connected with HPLC

Labeling by Tritium is generally achieved by either

reduction of unsaturated bonds with Tritium gas
(Tr) in the presence of Pd/C or Pd/BaSOo and or
tritiated reagents like NaBTo, LAI4, TMS3-SiT and

Nal

Analysis and Characterization

Analysis and characterization of

radiolabeled
compounds requires dedicated instruments. Structural
elucidation of the labeled compounds can be carried
out using dedicated instruments such as NMR, IR, UV

and LC-MS. Other specialized equipment includes

Liquid Scintillation Counter (LSC) for determining
the total and specific radioactivity of the labeled
compound. This technique involves mixing of the
known amount (weight or volume) of the sample
either in aqueous or organic scintillation liquid
and placing the vial in LSC instrument followed
by counting the radioactivity (disintegration) in
counts either DPS/DPM (Disintegrations per
Seconds/lvlinutes) or Counts Per Seconds/Minutes
(CPS/CPM). Counting efficiency forraC is -80-95%

DIBALT3 and 82T6 amongst others.l Further the

exchange of H, with T, gas using Crabtree's catalysts
or TrO in the presence of [Cp*(PMer)Ir(CO)(Et)J

Radiochemical purity

[C1]6 has been repofted.

analyzed using the radio Thin Layer Chromatography

and 40-45%o

for

3H.

of the labeled compound

is

Spinco Biotech

CuttingEdge
I

April 2014

scanner (e.g., Scan

RAM Radio-TLC)' In

this

technique, Glabeled compound (test sample) is spotted

(i) on ihe silica coated glass TLC plate (Fig-3C) and

iterelopea using appropriate solvent(s)' After drying'

for the
the plaie is placed on the scanner and measured
purity and intensity of the compounds (ii and iii) very
accuiately and the readout is obtained (Fig' 3B)' The
drawback however, being non-radioactive chemical
impurity can neither be detected nor quantified'

An altemative method, involves an online p-RAM

(Lablogic) detector connected to High Performance

-hromatography (HPLC) (Fig-a) which enables

as
simultaneous determination of the chemical as well
the
using
compound
labeled
the
of
purity
radiochemical
UV and/or ELSD detectors. In this method, on injection
the sample is separated in a column and the eluent
purr", ihtorgh the WELSD detector followed by
p-nu- detector. Before entering the p-RAM detector'
ihe sample mixes with the liquid scintillation cocktail
and the radioactivity is measured and chromatograph

iiquid

generated

in the system along with

UV/ELSD

Ihromatogram with a time lag of a few micro seconds

(Fig-s). Other instruments such as bench top counters
unJ tni"to plate readers are also available to estimate
the radioactivity in the biological samples'

While the analysis and characterizationof metabolites

in biological and test samples can be accomplished
by gpt-CA-fY HPLCA4S and HPLC/MS/MS another
highly accurate instrument called 'Accelerator Mass
Specirometry' (AMS) is also employed for

unique
often
methods
LCIMS/MS
and
applications. LC/MS
identification
structural
for
yieta amUiguous data

and exhaled air) and collected for identiflcation,

drug as well as
luantification, profilirrg of the parent
its metabolit"t. Th" total radioactivity is measured
using scintillation counters or radio detectors' While
theoretically, the complete mass balance recovery
should Ue iOO'1, of the administered radioactivity, in

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practice100o/orecoveryistechnica11ynotfeasib1eand
the current accepted recovery ranges from 85-900/o'7'8

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Currently, there is no wideiy ac-cepted and defined

cut-off value with a compelling underlying rationale
in this field. Different recovery ranges have been I
recorded in the literaturee for the recovery oflaC
labeled compounds in different species ranging from
82-106% lratq,le-rc2% (dogs), 58-94% (monkeys)
I

;
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and 61-105%

(humans)'

Drug metabolism

studres

The effect of different biological reactions on radio

labeled drug candidates are studied by evaluation of
blood and other physiological samples that have been
isolated as their metabolites after an administration

:
I

isotopically labeled drug' These metabolites

ure syntherired and separateiy evaluated for their

of

toxicity.
Whole Body

AutoradiograPhY

The Whole Body Autoradiography technique $fBA)

involves the intravenous injection of the radiolabeled
compound to a series of animals. At various intervals
these animals are rapidly frozen and sagiual microtome
sections are taken. These sections are further

freeze-dried,

followed by pressing against photographic X-Ray

Determination of the total dose administrated and

excreted (Mass Balance)

The radiolabeled molecule is administered to the

animal or human species and the total radiolabeled

material baiance is accounted for in the blood samples

(pharmacokinetics),

April 2014

in the

excreta (urine,

faeces

i
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data

andtimetoacquirethedata.Deve1opmentofsensitive
detectors like phosphorimagers to replace X-Ray fllms'
ability to produce the images of distribution/deposition
of radioactivity in various parts of the body so that it

Radiolabeled molecules flnd use as tracers in ADME,

pharmacokinetics and e-fate studies'

to estimate the radioactive

distribution in the tissue for example in

to get radiographic

materials, which are unavailable from in vlvo samples'

Based on AMS, after identification of the metabolite,
the synthesis of the metabolite is carried out to enable

Applications

film

compound
tumour and Blood Brain Barrier (BBB) penetrattons'
This technique requires more radioactive compound

detailed structural elucidation.

Lf metabolites, in situations where extremely low

concentrations of parent radiolabeled compound(s)

are used. These studies require more quantities of

can also be quantified as some of the salient features

of this technique. Currently Quantitative Whole Body
Autoradiography (QWBA) technique is used in animal

models.
e-Fate

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studies

Extensive use of agro and veterinary chemicals have

led to their dangerously high levels in the environment

ultimatelyimpactingliving orgqnisms' Hence,regulatory

authorities like U.S. Environmental ProtectionAgency
(EPA) and REACH (European countries) have placed
stringent restrictions on the usage of such chemicals
Spinco Biotech

CuttingEdge

l
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I

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u*,,*'ftf

anatrvttro candirare-s
.

:--;-s*;F

i'.-,

Many

I
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Drug

ti.,,,

iL

-I-#nn'^=

I
I

Path.Ai Convenlio"", rrnr*, *rr,"prnunt pr"grrrrr, [,.

rl*.'.A.!aI|veilUqnalrtsrlcelqevelopmenIprogramma:\

NCE \

The time taken to obtain human PK data ,or one

canbeupto

18-24moolhs

path-B Mrcrodosins deveropm*nrprosla,Tff'

Th6 tim6 tak6n to obtain human pK data for onelp.liie

would be approx 4

Reri /vafrre Reyre ws Drug Di$covery2, 2003,

W, *1 il4c.taboteodirgl
.Microdosng piiirmacotogicat Ooses rs p'reEAeO'
ir# microo'osing, in iitico', n-iirZ
^d"tr.
(Phase-o)

o) fr''"
l.t:
*..
_

NCE

rnonth$

Limited preclinical

23i

ff!,,i#*;l

,fn*",[i3;,o,,3, ,1f,3

:xf,:^,#3il
,jt
ryt Sry lsiding to. -40% atlritior

Toxicology

3;ffi*I3J,"",?j"r',*F,lj1l!li" r,l3
#iiion irt" o*-ig tJ p.oii,x *,ri ue

ffi'

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and have mandated chemical developers

to

discovery programs. Currently, while several mid-sizec

drug discovery and biotech companies focus on expanding
their pipeline (new drug) developments, catering to the

Bio-degradation, Bio-concentration (persist in water or

sediment), plant and animal metabolism (accumulation
in tissues of animals/plants) etc. To evaluate the fate of
the chemical, the parent compound is laC labeled and

radiolabeling requirements have opened new avenues

for CROs to offer this unique and differentiated service
the market. Over a year ago, GVK Biosciences private
Limited, in Hyderabad started offering this service by

used as tracers.

setting up one of the best equipped facilities in India which

has also been approved by the Atomic Energy Regulatoq'

Microdosing

Board (AERB), Govemment of India.

to determine the fate of the chemical after

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Microdosing also called 'phase-0' or 'flrst in human'

study and before clinical phase-I studies is a relatively
new technique that has received considerable attention of
all stakeholders in drug development cycle. It is defined
as 100 fold less than (1/100'h) the pharmacological dose
(determined using animal models and in vitro systems)
and should not exceed 100 pg, co-administered with raC
labeled drug (50-500 nano Curies)10 in healthy volunteers

for early

characterization

of PK and

distribution

properties using Accelerator Mass Spectrometry (AMS).

The advantages of this technique include the absence
of biological effect and reduced risk potential to human
volunteers. AMS being very sensitive, enables measuring
the quantity of isotope (especially raC) in parts per
quadrillion (ppq) levels.

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This technique has been provisionally endorsed by FDA11

and European Medicines Agency (EMA). A consortium
is cur:rently working on this project known as 'Xceleron'

(www.xceleron.com) has recently observed that it was

involved in 4 of the 5 new medicines approved by FDA

in2013.
Spinco Biotech

Until recently most pharmaceutic al organizations har

in-house isotope labeling facilities to support their drug

conduct
its
release into the environment, either from an intended use
or an accidental release, before registration. This study
referred to as 'Environmental fate' studies, includes

studies

CuttingEdge

Conclusion
Radiolabeled molecules or their metabolites are 'GOLD
STANDARDS' to establish pharmacokinetics, ADME.
toxicity and environmental (soil and water) fate studies.
Microdosing of radiolabeled drugs also now offers the
prospect of the oppornrnity of working directly into

human studies. Therefore radiolabeled compounds

continue to play a significant role in new molecule
development programmes.

References
l2.
3.
4.
5.
6.
7.
8.
9.

RoA foges, J. Richdrd Heys, Thomu Moenius, Preparation oJ Compounds Labeled with
Trliuil aild Cdrbon-ll, Wilql (2009)
SfiniyasG,UnnyVK,MukkantiK,ChoudatyBM,ApptRadiatlsot.20t3,.Z2:145-51
M. F Bdrakd. A. N. Farag, M. T. Ragab, M. M. El-Fouly & F K. Et-Baz , Isotopes in
Lnv;ronnental aal Health Stddi,,. I 9q0. - | 2q. 3 I t - 3 I R. Swanson d O. Hoegh-Guldberg, Marine Biolog), 1998, 131, 83 93; J. T. Wang, A. E.
Dougld, Marine Biolag, 1999, 135,2, 219-222
Ctdbtree,R.H.(1979)."lridiumcompoundsincatab)sis".Acc.Cheil.Resj2(g):331-337,
J. Ory. Chem. 1936,51 (14):2655 2661.
Beryman, R. G., J. Amd. Chm. Soc., 2A02, 124(t0),2092.
Beunea J. H., Beilhen,.I- H., S.hellens, J. H. M. Mass baldnce studies, with a focus on
anticancer drugs. Clin. Pharnacokiilel2006, 15.33 58
Suwel, M., C.G. Sahajwalla, Netr Drug Development, Regulatary paradigils Jbr Ctinicat
Pharamcology and Biopharmaceutics. New York: Marcel Decke4 2004, pp. 1A7-2j2
Sarah J. RolJLy, R. Scott Obach, Jenny 1- Gedge, and Dennis A. Smith, Dlug Metabolism

Revietts, 2007, 39: 1743

10. Brend Oosterhuis, Bioanalysis,

201A, 2(3), 377-379.

11. Guidance _for lndust!, Inrestigdtors, dnd Reviewers. Etploratary IND Stodies. FDA,
CDER, Jantnry 20a6

ETA

April20L4