Radiotracer Applications in Biologics - Indian Pharmaceutical ...

Article
Radiotracer Applications in Biologics
N. Sivaprasad*
Board of Radiation and Isotope Technology, Dept of Atomic Energy, BARC Valhi Complex, Navi Mumbai
Introduction
Biopharmaceutical science and biotechnology have been finding
major therapeutic solution, through biologics, to many diseases.
In fact, currently few biomolecules and biologics based medicines
have emerged to help lakhs of patients all over the world. Biologics
are expected to capture a good share in drug market to the extent of
about 40% in the near future. But these drugs are very complex in their
structure and behavior and need research tools for their evaluation
and understanding. Radiotracer techniques along with nuclear
imaging are emerging as important research tools for biologics. This
article highlights some of the applications of radiotracers.
Radiotracer is molecule having one or more atoms of radioactive
isotopes and thereby radiotracers are able to emit radiations such
as alpha particles, beta particles, gamma rays, positron etc. due to
the spontaneous disintegration of the radioisotopes present in the
radiotracer. Chemically and thus biologically the radiotracer behaves
in similar manner as that of the non-radioactive counterpart. In fact,
the basic principal behind the use of radiotracer in research and
investigation is that all the isotopes, both radioactive and stable,
of a given element will chemically behave similarly. Thus, atoms of
125
I –a radioactive isotope of iodine-will behave same as that of the
stable isotope of iodine, 127 I in any chemical and biological system. It
is easy to detect the radiotracer than its stable counterpart and thus
this provides a good research and investigational tool in biological
systems. In addition the sensitivity of detection of a radiotracer is
higher than that of the inactive one. For example, in any chemical
technique detection sensitivity achievable is about 10 13 to 10 15
atoms for observing a chemical response, whereas, in the case
of radioactivity, it is theoretically possible to detect few number of
atoms to the extent of an analytical sensitivity of 10 4 to 10 5 folds
. This higher sensitivity, enables the detection of very low levels
of analytes, for example identification of those metabolites which
otherwise miss the detection in conventional analytical methods.
Radiotracer has been in use as research tool in the very early
development of biopharmaceuticals and biologics. Biologics are
medicinal products derived from biological processes. Vaccine,
blood components, antibodies, RNAs, stem cells that are
pharmacologically and pharmaceutically safe for human use are
classified as biologics in general and have application as drugs for
the treatment of various diseases. Initially, in around 1998 this class
of drugs called biologics based on biological biomolecules became
an active area of pharmaceutical research. Their mechanism of
action is general based on the biological interaction leading to a
physiological/biological response. For example, the drug action of
antibody depends on the antigen and antibody binding reaction,
RNA or DNA depends on either hybridization with complementary
DNA site or induction of protein expression, the vaccines on immune
response etc. Hence, the effectiveness of these drugs is generally
based on the potency & retention of their biological property and
ability to elicit biological response. Their potency and efficacy also
depends on their biological integrity, function & interaction with other
biomoleules. Using a radiotracer of either the interacting molecules
(biologics) or the ones responding to the interaction, it is possible
to understand their potency, efficiency and the dosimetry (tissue
distribution) in a complex biological system. It is possible to locate
the site of interaction as well as to map the presence and path of
the molecules of biologics in a biological system.
Consider the following example; for the intracellular signal
transduction, the enzyme Phosphodiesterase (PDE) plays an
important role by regulating the neurotransmission–the process by
which signaling molecules called neurotransmitters are released by
a neuron- that interact and activate the receptors of another neuron.
Phosphodiesterases are a diverse family of enzymes that hydrolyze
cyclic nucleotides and thus, regulate intracellular levels of the second
messengers cAMP and cGMP, and hence the cell functions. The
distribution & expression of this enzyme can be mapped by imaging
using a specific radiotracer ligand/substrate of these enzymes. The
radiotracer imaging is expected to allow a better understanding
of physiological and pathological processes related to enzyme
expression and function in the brain. 18 F-labeled enzyme ligands are
being investigated for mapping the enzyme using Positron Emission
Tomography (PET). The radiotracers thus, can also be of use in the
study of drugs based on inhibition of phosphodiesterase
Radiotracers of Neurotransmitters and their receptors
Investigating the neurotransmitter receptors and peptide
hormone receptors which act as specific target molecules for
targeting of tumors and investigating drugs for neurological & psychiatric
disorders is an established and also one of the important current areas
of interest to pharmaceutical scientists. For example, it has been in
research to use the somatostatin peptide analogues as drug for the
treatment of PLCD (Poly Cystic Liver Disease). The peptide somatostatin
is a Growth hormone (GH) inhibiting hormone that regulates the
endocrine systems and affects neurotransmission and cell proliferation
via binding to G-protein-coupled somatostatin receptors resulting in the
inhibition of the release of many secondary hormones. Somatostatin
analogs labelled with 131 I (Iodine -131) or 99m Tc (Technitium-99m) can
thus be used to image the sites of binding of the drugs and also it can
provide quantitative information on retention and elimination of the drugs.
Such radiotracer will be an excellent tool in animal studies on designing
somatostatin peptide analog based drugs
Radiolabelled Somatostatin Analog
Another area of direct and important application of radiotracers
of somatostatin analog is in the identification and detection of
nueroendocrine tumors (NET) by nuclear imaging, that is by
determining the distribution of the radioactivity in the tissue. The
basic principle behind this diagnosis by nuclear imaging is that
the somatostatin receptor is over expressed on a wide variety of
nuro endocrine tumors (NET). The somatostatin analog, octreotide
labeled with 111 In (Indium-111) or 99m Tc (Technetium-99m) can bind
to the receptors and is the standard procedure for the diagnosis
of NET by nuclear imaging. Octerotide is an octa-peptide that
mimics the natural somatostatin pharmacologically and is also
used as a peptide drug in the treatment of acromegaly, gigantism,
thyrotropinoma, and diarrhea in patients with vasoactive intestinal
peptide (VIP) secreting tumors. Several octreotide derivatives
labelled with a variety of radionuclides such as 111 In, 99m Tc , 131 I and
*Email Id: shiv2011@rediffmail.com
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90
Y(Ytterbium-90) have been successfully used for imaging and
also for radiotherapy of NET. The major benefit of peptides that
are chemically tagged with a radio metal such as 90 Y, 99m Tc or 111 In
is the build–up of the radio-metal-complex in the target cells. Thus
this enables the specific detection of tumor site and their metastasis
as well as delivery of radiation at the tumor site enabling the
treatment. When the peptide carrying radio metal is internalized, the
radiolabelled metabolites are trapped in the lysosome and they can
be identified. As mentioned earlier, labelled ocreotide peptide can
hence be a research tool in the development of receptor based drugs
and biologics for NET. Another important example of neurotransmitter
receptor targeting is based on the use of dopamine receptor.
Radioisotope tagged dopamine derivatives are commonly used to
target tissue that over express the dopamine receptor. These tracers
are clinically used to diagnose dementia and some extent Parkinson’s
disease (PD). The tracers are also used to study dementia and drug
addiction. The radiotracer study can be used to correlate to the degree
of dementia – a supporting aid for the quantitation of the efficacy of
the drug and biologics for dementia treatment
Peptide & protein based radiotracers
Other than somatostatin analogs and neurotransmitters, there
are many peptide based radiotracers are being studied for nuclear
medicine and imaging applications. These are pharmaceutical
formulations of radiotracers of peptides. Peptide based radiotracers
are potential imaging agents for understanding brain disorders,
should these agents be enabled to undergo transport through the
blood-brain barrier (BBB) in vivo. Radiolabeled Amyloid beta–protein
is a radiotracer of protein based biologics to image and detects brain
amyloid in tissue sections of Alzheimer’s diseased autopsy brain. The
13I
I labelled protein degrades in brain with release of radioiodine 131 I.
Radiolabelled amyloid-beta peptide (Abeta) has been shown to label
neuritic plaques (extracellular deposits of amyloid in the gray matter
of the brain and the deposits are associated with degenerative neural
structures) in vitro, therefore it could provide a detection tool, if it also
labels neuritic plaques in vivo following intravenous injection. It has
also been shown that the Abeta possess permeability at the bloodbrain
barrier. In conclusion, these studies describe a methodology for
evaluation of BBB drug delivery and targeting of peptides in brain.
Radiotrace for cell proliferation studies.
For in-vitro investigation studies of drugs and biologics, cell
culture technique is one of methods, where the determination of
viability and cell proliferation is required. For investigation of cancer
drugs testing in cancer cell culture and studies on cell proliferation is
an established in-vitro procedure. One of the most familiar and widely
used methods for quantifying cell proliferation is the measurement of
tritiated thymidine ([ 3 H]-thymidine) incorporation. Cells incorporate the
radiolabeled DNA precursors into newly synthesized DNA, such that
the amount of incorporation, measured by liquid scintillation counting,
is a relative measure of cellular proliferation.
Endothalial cell proliferation has been studied by radioligandreceptor
binding. The principal is that the ligands that bind to sigma
receptors can modulate the proliferation of the endothelial cells,
which can then control the angiogenesis. Thus, these receptors
have become a potential target for application in oncology. Other
areas of interest for investigation are cardiovascular and endocrine
system disorders. Some of the steroids such as progesterone,
testosterone and dehydroepiandrosterone (DHEA), interact with
sigma-1 receptors. Radiolabelled steroids with 99m Tc or 131 I can then
be of help for development of receptor ligand analog.
Radioligands for sigma receptor
Sigma receptors have also a role in manifestation of certain
Alzheimer’s related disease. The precise biological functions
of sigma receptors have not been well established. Recent
investigation using radiotracers of the receptor binding ligands has
shown that Sigma receptor has the potential of being a target for
development of biologics related to psychiatric and neurological
disorders. Sigma receptors are classified into sigma (1) and sigma
(2) subtypes. There are attempts in the development of radiotracer
for imaging sigma -2 receptors so as to have a research probe.
This approach is based on the experimental evidence that the
sigma-2 receptors are expressed in high density in a number of
human tumors, including breast tumors. Preliminary data so far
obtained indicates that radioiodine labeled sigma-2 receptor binding
radiotracers can not only be used in the anatomical imaging of
breast tumors, but may also provide information regarding the
proliferative status of the breast cancer. Any biologics or drugs that
is designed for inhibiting the proliferation of the cells as a measure of
inhibiting the growth of the tumor can be investigated by estimating
their effect of the drug on the proliferation of the cells using such
radiotracer both by in-vivo as well as in-vitro procedures. For this
and other reasons, sigma-1 receptors have been proposed as a link
between the central nervous system and the endocrine & reproductive
system. This is largely an important, but an unexplored area. Thus, a
promising area of research is to examine the biological function and
therapeutic potential of sigma receptors which can be achieved through
radiotracers. Subtype of sigma receptor has distinct physiological and
pharmacological profile in the central and peripheral nervous system.
The characterization of these subtypes and the discovery of new
specific sigma receptor ligands through radiotracer demonstrated that
sigma receptors are targets for the development of suitable biologics
for treatment of neuropsychiatric diseases such as schizophrenia and
depression. Furthermore, considering the potential of sigma receptor
investigation, imaging of sigma (1) receptors in brain using specific
PET radio labelled ligands have been initiated recently.
P-glycoprotein
P-glycoprotein (P-gp) is associated with cell membrane. P-gp
is a well-characterized transporter protein. This protein is encoded
by multidrug resistant gene (MDR1). P-gp transports number
of molecules across extra- and intra-cellular membranes. This
protein pays a role in the removal of drugs from the intracellular
compartment of the cells. Thus this causes a decrease in drug
build-up in drug-resistant cells and often mediates the development
of resistance for drugs for cancer treatment. In the case of BBB
this protein also functions as a transporter. The radiotracers of
high specificity and affinity inhibitors of P-gp are shown to be a
good biologic marker to assess the expression and distribution
of this transporter. Radioactive [ 11 C]-verapamil has been used
for measuring P-glycoprotein function with positron emission
tomography (PET). Verapmil is an L-type calcium channel blocker of
the phenyl alkylamine class. Under conditions where the expression
of P-gp is increased, the intensity of PET signal also correspondingly
increases. At the same time, the uptake of substrates of the
transporter decreases. Studies on binding of other radiotracers such
as [ 11 C] laniquidar with PET can also be of use for assessing P-gp
expression. Laniquidar is an inhibitor of P-gp. The technique and
application of PET is described elsewhere in this article.
Stem cells
Stem cell is cellular biologics. Animal studies and clinical trials
have shown that there is a potential with stem cell based therapy
particularly for the cardiovascular diseases. Stem cells are now
attempted to treat conditions such as leukemia and have the potential
to treat many more diseases and disorders where patient survival is
reliant on organ and tissue donation. Stem cell research and its clinical
applications are expected to make the stem cell a regular biological
drug for therapy in future. The cardiovascular stem cell based therapies
have been established fairly beyond doubt. Radiotracer can also play
a role in the determination of the fate of stem cell non-invasively in a
biological system. The most important in the stem cell therapy is the
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evaluation of the implanted cells. Currently, it is difficult to establish
whether stem cells have survived following transplantation in the body
and if they reach their target site or migrate elsewhere. Radiotracer
based PET and SPECT (Single Photon Computed Tomography) are
recommended to be one of the effective means to track the cell lines.
The radiotracer for this study can either be done by direct labeling of
the stem cells or through reporter gene. In the direct methodology, the
stem cells are radiolabelled and followed for few hours. The cell labeling
methods are well established. White blood cells are labelled using the
labelling agent [ 99m Tc] hexamethyl propylene amine oxime (HMPAO) for
imaging inflammation site. Similar method can be adopted for labelling
stem cells. The labelling involves a short incubation with the labelling
agents and subsequent purification. Whereas, in the second method,
the stem cells are transfected with reporter gene and the expression
is imaged by a suitable radio substrate that can bind to the gene
expressed molecules, thereby the assessment of cell survival and
proliferation can be monitored non-inversely and externally.
PET Imaging
Nuclear Imaging is an excellent tool for understanding the tissue
distribution of biologics and their movement in a living system noninversely.
The biomolecules under investigation can be radiolabelled
with either gamma emitting or positron emitting radioisotope to
prepare a radiotracer. The radiotracer is administered to human
or animal internally, for example intravenously or orally. Then,
external detectors (Gamma or PET camera) capture the radiation
and form images from the radiation emitted by the radiotracer.
This process is unlike a diagnostic X-ray where external radiation
is passed through the body to form an image. PET and SPECT
are techniques used for this purpose. PET uses positron emitting
radiotracer. In the case of positron emitters the positron undergoes
annihilation with electrons and gamma rays are generated which
are detected by PET camera and constructs the images. SPECT
uses gamma emitting radiotracers.
PET and SPECT based nuclear imaging can detect the
radionuclide tagged to biologics which interact with biochemical
process in living subjects. This imaging modality is very sensitive to
the extent of 10 -11 moles of radiotracer, this sensitivity enables PET and
SPECT to detect the biological process in a very low levels. Because
of the sensitivity this imaging techniques can offer, the radiotracer that
needs to be administered is also very small, that it does not alter the
biological equilibrium of the biochemical reactions and pathways. PET
and SPECT though initially developed for imaging in human, now the
technology offers imaging in small animals enabling to carry out the
studies in small animals such as mice, rat etc. The special imaging
resolution can also be as slow as 3-2 mm.
In fact human in vivo nuclear imaging by PET brought a new
concept in pharmacology, and it can answer many questions
related to drug development. Biodistribution studies with biological
molecules carrying positron-emitting radioisotopes can test whether
a bioactive molecule reaches a target tissue compartment (such
as brain) in adequate amounts so as to effect the pharmacological
activity. Combined application of PET and Magnetic Resonance
Imaging (MRI) modality allows the understanding of the relation of
molecular interactions with anatomical or physiological changes
in tissue. Recently the drug researchers are of the opinion that
the potential value of nuclear imaging should be considered at
the earliest stages of development of drugs as PET allows the
determination of the effect of a biologics on patients’ metabolic
parameters. In addition, PET may be used to follow the fate of a
compound in vivo, allowing visualization of its binding to specific
receptors and a direct study of the mechanism of drug action in
normal and pathological situations.
Structural information is considered to be essential for
determining the protein binding with molecules in the target
tissues. Currently the focus is screening of the multiple biologic
target and rational design of high-affinity peptide phamachore for
the development of biologics using large throughput assays. This
type of approach is required for characterizing the large number of
proteins currently being generated by structural genomics research.
Radiotracer based PET can play a significant role in development of
such biologics. In clinical trials, it is possible to test whether it retains
the biological potential, whether the biologic molecules reached the
target tissue. If so what would be the effective quantity available
at the target site etc. A radiolabelled biologic with 11 C or 18 F and
with the aid of PET can throw light on these questions. Another
area of application of PET is binding studies with a radiolabelled
analog of the bioactive moiety of biologics that binds to the target
of therapeutic interest with adequate specificity and affinity. This
will enable direct assessment of the relationship between biologics
blood concentration and target occupancy. Such radiotracers can
be of use in quantitating relative rates of biological processes,
which is useful pharmacodynamic information. Apart from this PET
and SPECT can detect and quantitate physiological processes
— such as tumor activity, brain function etc. The nuclear imaging
using radiolabelled pharamcore of the biologics can thus, reveal
the pathophysiology of disease; tumor metabolism and tumorassociated
angiogenesis provided the radiolabelled biomolecule
entity is stable in physiological conditions.
Quantitative whole-body autoradiography (QWBA)
QWBA is an in-vivo radiotracer technique carried out in
animals and dissected human tissue, which has evolved as one
of the important radiotracer studies in drug development. Whole
body autoradiography produces an image of the distribution of a
radioactively labelled test drug and biologics over the entire animal
body sections. After administration of radiolabelled tracer the
animal is euthanized and snap frozen. Frozen sections are cut and
radioactivity distribution is imaged. Radioactivity levels in individual
structures are quantified and results are expressed as amount of
drug equivalent per unit mass of tissue. In these studies, 14 C or 125 I
labeled drugs and biologics are commonly employed for the study
of tissue distribution and pharmacokinetics. Furthermore, the use of
radiolabelled standards and digital image processing can give more
accurate qualitative and quantitative analysis in whole-body sections.
QWBA is the preferred method for tissue distribution studies required
for regulatory filing of a new drug entity and for projecting tissue
dosimetry in human mass balance studies. This commonly used for
drugs is very well applicable for biologics also.
Conclusion
When it comes to biologics stringent evaluation and characterization
is very important and essential. For example stringent purity and
clinical safety requirements are established by regulatory authorities.
Also the cost of development of new biologics has been phenomenal
and concerted efforts are necessary to address cost reductions.
The use of radiolabelled biomolecules and related techniques has
recently shown great promise as has been demonstrated by various
researchers. Due to the noteworthy impact of radiotracer based
studies and nuclear imaging in development of biologics and drugs,
clinical evaluation studies can be now performed earlier than that in
the past. Radiotracer techniques and nuclear imaging will definitely
benefit the research in the development of biologics
Further Reading
1. Jayachandran, N, Unny V. K. P, Sivaprasad N.
2. Radiolabelled compounds production Program in India.
3. Proc. Ninth Int. Symp. on Synthesis and Applications of Isotopically
Labelled Compounds’, Edinburgh, 16-20 July, 2006.
4. The World Health Organization (WHO)
5. Use of Ionizing Radiation and Radionuclide on Human Beings for
Medical Research, Training and Non-medical Purposes,
6. WHO Technical Report Series 611, Geneva: WHO, 1977
7. Graham Lappin & Simon Temple (Editors)
8. ‘Radiotracers in Drug Development’
9. CRC Press; 1 edition (2006)
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