PRINCE SATTAM BIN ABDUL AZIZ UNIVERSITY
COLLEGE OF PHARMACY
Nuclear Pharmacy
(PHT 433 )
Dr. Shahid Jamil
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Radiolabeling
 The use of compounds labeled with radionuclides
has grown considerably in medical, biochemical,
and other related fields.
 In the medical field, compounds labeled with β- -emitting
radionuclides are mainly restricted to in vitro experiments
and therapeutic treatment, whereas those labeled with
γ
-emitting radionuclides have much wider applications for
in vivo imaging of different organs.
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In a radiolabeled compound, atoms or groups of atoms of
a molecule are substituted by similar or different
radioactive atoms or groups of atoms.

In any labeling process, a variety
of physicochemical conditions can
be employed to achieve a specific
kind of labeling.
 There
are
six
major
methods
employed in the preparation of
labeled compounds for clinical use.
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General methods of radiolabeling.
Isotope Exchange Reactions
• In isotope exchange reactions, one or more
atoms in a molecule are replaced by isotopes
of the same element having different mass numbers.
• Since the radiolabeled and parent molecules are identical
except for the isotope effect, they are expected to have
the same biologic and chemical properties.
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These labeling reactions are reversible and are useful for
labeling
iodine-containing
material
with
iodine
radioisotopes and for labeling many compounds with
tritium.
Examples:
125I-labeled
triiodothyronine (T3),
125I-labeled
thyroxine (T4),
14C-, 32S-,
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and 3H-labeled compounds.
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Introduction of a Foreign Label

In this type of labeling, a radionuclide is incorporated
into a molecule that has a known biologic role, primarily
by the formation of covalent or coordinate covalent
bonds.

The tagging radionuclide is foreign to the molecule and
does not label it by the exchange of one of its isotopes.
Some examples are
51Cr-labeled
red
99mTc-labeled
blood
cells,
albumin,
and
99mTc-DTPA,
many
iodinated
proteins and enzymes.
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
In several examples, the in vivo stability of the material
becomes uncertain and one should be cautious about
any alteration in the chemical and biologic properties of
the labeled compound.

In some instances, a chemically analogous radionuclide
can be substituted for an atom already present in the
molecule; for example,
75Se
can replace sulfur in
methionine to form 75Se-selenomethionine.
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
In many compounds of this category, the chemical
bond is formed by chelation, that is, more than one
atom donates a pair of electrons to the foreign acceptor
atom, which is usually a transition metal.

Most of the
99mTc-labeled
compounds used in nuclear
medicine are formed by chelation.
 Example, 99mTc binds to DTPA,
gluceptate, and other ligands by chelation.
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Labeling with Bifunctional Chelates
 Bifunctional
chelates
such
as
EDTA,
DTPA,
and
desferoxamine have been used successfully in the labeling
of various proteins.
 In
this method, proteins are allowed to form complexes
with the bifunctional chelating agent and the complex is
then labeled by chelation with an appropriate radionuclide.
 Examples:
111In-labeled
DTPA-albumin,
67 Ga-labeled
desferoxamine-albumin,
99mTc-labeled
 Because
DTPA-antibody.
of the presence of the chelate, the biological
properties of the labeled protein may be altered and must
be assessed before clinical use.
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Biosynthesis
In biosynthesis, a living organism is grown in a culture
medium containing the radioactive tracer.
The tracer is incorporated into metabolites produced by
the metabolic processes of the organism, and the
metabolites are then chemically separated.
Example, vitamin B12 is labeled with
6OCo
or
57Co
by
adding the tracer to a culture medium in which the
organism Streptomyces griseus is grown.
Other examples,
14C-labeled
carbohydrates, proteins
and fats, and L-75Se-selenomethionine.
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Recoil Labeling
Recoil (retreat) labeling is of limited interest because it
is not used on a large scale for labeling.
In a nuclear reaction, when particles are emitted from a
nucleus, recoil atoms or ions are produced that can form
a bond with other molecules present in the target
material.
The high energy of the recoil atoms results in poor yield
and thus a low specific activity of the labeled product.
Several tritiated compounds can be prepared in the
reactor by the 6Li (n, α) 3H reaction.
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The compound to be labeled is mixed with a lithium salt
and irradiated in the reactor.
Tritium produced in this reaction will label the compound
by the isotope exchange mechanism and then the
labeled compound is separated.
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Excitation Labeling
Excitation labeling entails the utilization of radioactive
and highly reactive daughter ions produced in a nuclear
decay process.
During β decay or electron capture, energetic charged
ions are produced that are capable of labeling various
compounds of interest.
Krypton-77 decays to
labeled is exposed to
77Br
77Kr,
and, if the compound to be
then energetic
77Br
ions label the
compound to form the brominated compound. Various
proteins have been iodinated with 123I by exposing them
to
123Xe,
which decays to 123I.
The yield is low with thisL8-method.
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Isotope exchange
125I-labeled
T3 and T4
14C-, 32S- and 3H-labeled compounds
Introduction of a
foreign label
All 99mTc radiopharmaceuticals
125I-labeled proteins
125I-labeled hormones
111In-labeled cells
18F-fluorodeoxyglucose
Labeling with
bifunctional chelates
111In-DTPA-albumin
Biosynthesis
75Se-selenomethionine
99mTc-DTPA-
antibody
57Co-cyanocobalamin
14C-labeled
Recoil labeling
3H-labeled
Excitation labeling
123I-
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compounds
compounds
Iodinated compounds
labeled compounds (from 123Xe
decay)
77Br- labeled compounds (from 77Kr decay)
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Important Factors in Labeling
The majority of radiopharmaceuticals used in
clinical practice are relatively easy to prepare
in ionic, colloidal, macroaggregated, or
chelated forms.
Many of them can be made using commercially
available kits.
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Several factors that influence the integrity of
labeled compounds are:
Efficiency of the Labeling Process
Chemical Stability of the Product
Denaturation or Alteration
Isotope Effect
Carrier-Free or No-Carrier-Added (NCA) State
Storage Conditions
Radiolysis
Purification and Analysis
Shelf Life
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Efficiency of the Labeling Process
A high labeling yield is always desirable, although it
may not be possible in many cases.
The higher the yield, the better the method of
labeling. But a lower yield is sometimes acceptable if
the product is pure and not damaged by the labeling
method, the expense involved is minimal, and no better
method of labeling is available.
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Chemical Stability of the Product
Stability is related to the type of bond between
the radionuclide and the compound.
Compounds with covalent bonds are relatively
stable under various physicochemical conditions.
The stability constant of the labeled product should
be large for greater stability.
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Denaturation or Alteration
The structure and/or the biologic properties of
a labeled compound can be altered by various
physicochemical
conditions
during
a
labeling
procedure.
Example, proteins are denatured by heating, at
pH below 2 and above 10, and by excessive
iodination.
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Red blood cells are denatured
by heating.
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Isotope Effect
The isotope effect results in different physical and
biologic properties due to differences in isotope weights.
Example:
In tritiated compounds, H atoms are replaced by
3H
atoms and the difference in mass numbers of 3H and H
may alter the property of the labeled compounds.
Also the physiologic behavior of tritiated water is
different from that of normal water in the body.
The isotope effect is not serious when the isotopes are
heavier.
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Carrier-Free or No-Carrier-Added (NCA) State
Radiopharmaceuticals tend to be adsorbed on glassware if
they are in a carrier-free or NCA state.
The molar concentration of carrier-free compounds is in the
range of nanomolar or less, and it is very difficult to study
their chemical behavior in such a low concentration.
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Storage Conditions
o
o
o
o
Many
labeled
compounds
are
susceptible
to
decomposition at higher temperatures.
Proteins and labeled dyes are degraded by heat and
therefore should be stored at proper temperatures;
Example:
Albumin should be stored under refrigeration.
Light may also break down some labeled compounds
such as radioiodinated rose bengal thus should be stored
o
in the dark.
The loss of carrier-free tracers by adsorption on the
walls of the container can be prevented by the use of
silicon-coated vials.
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Radiolysis

Many labeled compounds are decomposed by radiations
emitted by the radionuclides present in them.


This kind of decomposition is called radiolysis.
The higher the specific activity, the greater the effect of
radiolysis.

When the chemical bond breaks down by radiations from
its own molecule, the process is termed "autoradiolysis".
 Radiations
may also decompose the solvent, producing
free radicals that can break down the chemical bond of
the labeled compounds; this process is indirect radiolysis.
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
Example:
radiations
from
a
labeled
molecule
can
decompose water to produce hydrogen peroxide or
perhydroxyl free radical, which then oxidizes another
labeled molecule.

To prevent indirect radiolysis, the pH of the solvent
should be neutral because more reactions of this nature
can occur at alkaline or acidic pH.

The longer the half-life of the radionuclide, the more
extensive is the radiolysis,
and the more energetic the radiations, the greater is the
radiolysis.
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
Radiolysis
introduces
a
number
of
radiochemical
impurities in the labeled material and one should be
cautious about these unwanted products.

These factors set the guidelines for the expiration date
of a radiopharmaceutical.
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Purification and Analysis
Radionuclide
impurities
are
radioactive
contaminants
arising from the method of production of radionuclides.
Fission method produce more impurities than nuclear
reactions in a cyclotron or reactor because there are
numerous modes of fission of the heavy nuclei.
Target
impurities
also
add
to
the
radionuclidic
contaminants.
Radiochemical
and
chemical
impurities
arise
from
incomplete labeling of compounds.
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
Often these impurities arise after
labeling from natural degradation
as well as from radiolysis.
 Radionuclide
impurities can be estimated by various
analytical methods such as solvent extraction, ion
exchange, paper, gel, or thin-layer chromatography, and
electrophoresis.
 The
removal
of
radioactive
contaminants
can
be
accomplished by various chemical separation methods,
usually at the radionuclide production stage.
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Shelf Life
A labeled compound has a shelf life during which it can be
used safely for intended purpose.
The loss of efficacy of a labeled compound over a period of
time may result from radiolysis and depends on:
 The physical half-life of the radionuclide.
 The solvent
 Additives
 The labeled molecule
 The nature of emitted radiations
The nature of the chemical bond between the
radionuclide, and the labeled
compound.
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Usually a period of three half-lives or a maximum of 6
months is suggested as the limit for the shelf life of a
labeled compound.
The shelf-life of
99mTc-compounds
varies between 0.5 and
18 hr.
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