Radiotracers in pharmacy

Introduction ans applications of radio tracers in pharmaceutical field, techniques used for radio tracer imaging.

1. RADIOTRACERS IN PHARMACY Submitted by: Viraj Sukthankar Dept. of Pharmaceutics KLE college of Pharmacy Belagavi -Karnataka. KLEU'S College of Pharmacy 1

2. CONTENTS: Introduction Preparation and Characterization of radiotracers Applications of radiotracers Nuclear Imaging Techniques Marketed Products Patents Future scope Conclusion and References. KLEU'S College of Pharmacy 2

3. Introduction: A radioactive tracer, or radioactive label, is a chemical compound in which one or more atoms have been replaced by a radioisotope so by virtue of its radioactive decay it can be used to explore the mechanism of chemical reactions by tracing the path that the radioisotope follows from reactants to products. Radiolabeling is thus the radioactive form of isotopic labeling KLEU'S College of Pharmacy 3

4. Radioisotopes of hydrogen, carbon, phosphorus, Sulphur, and iodine have been used extensively to trace the path of biochemical reactions. A radioactive tracer can also be used to track the distribution of a substance within a natural system such as a cell or tissue or as a flow tracer to track fluid flow. Radioactive tracers are also used to determine the location of fractures created by hydraulic fracturing in natural gas production. Radioactive tracers form the basis of a variety of imaging systems, such as, PET scans, SPECT scans and technetium scans KLEU'S College of Pharmacy 4

5. Principle: The basic principle of the radiotracer is that the radioactive tracer will have the same chemical and biochemical properties as that of its non-radioactive counterpart, as the chemical and biochemical properties are governed in both tracer as well as in the non-radioactive entity by their reactivity which are depending only on the number of extra nuclear electrons and their organization in the electronic orbits. KLEU'S College of Pharmacy 5

6. Classifica tion: Uniformly Labelled (U): Uniformly labeled compounds are labeled in all positions in a uniform or nearly uniform pattern . E.g. L-Valine- 14 C (U) implies that all carbon atoms in L-valine are labeled with equal amounts of 14 C. Specifically Labelled (S): Chemicals are designated as specifically labeled when all labeled positions are included in the name of the compound and 95% or more of the radioactivity of the compound is at these positions . E.g. Aldosterone1,2- 3 H implies that <95% of tritium label is in position 1 and 2. Randomly Labelled or Generally Labelled (G): This designation is for compounds in which there is random distribution of labelled atoms in the molecule. Not all positions in a molecule are necessarily labeled . Nominally Labelled (N): This designation means that some part of the label is at a specific position in the material but no further information is available as to the extent of labelling at other positions . E.g. Cholestrol-7- 3 H (N), some tritium is at position 7, but may also be at other positions. KLEU'S College of Pharmacy 6

7. Advantages: The chemical behavior of radiotracers is not altered. Radiotracers can be quantified in minute quantities with high sensitivity. The detection instruments are inexpensive and easily available. Detection is fast and reliable. Sample preparation is easier and in many cases the radiotracers need not be isolated . Disadvantages: Nuclear/Radiation safety. Requirement of further chemical processing to determine the mass of material isolated and the specific activity. KLEU'S College of Pharmacy 7

8. There are two ways in which radiotracers are used When a radiotracer undergoes chemical reactions one or more of chemical products will be generated containing the radioactive label. The analysis of the radioactivity will be able to provide detailed information on the mechanism of such chemical reactions and thus enable in vitro biological investigations in biological systems . A radioactive compound (radiotracer) is administered into a living organism and the administered radioisotope provides a means to construct an image showing the way in which that compound and its reaction products are distributed around the organism. This image is generally known as scintigraphy. KLEU'S College of Pharmacy 8

9. Criteria for selection of tracers: In order to have meaningful results from a set of radiotracer experiments, it is very important that an appropriate radiotracer methodology is chosen based on certain parameters such as: Nature of the radiotracer. Nature of radiation. Specific activity of the radiotracer. Half-life of the radioisotope. Radio purity. KLEU'S College of Pharmacy 9

10. Preparation and characterization of radiotracers: There are four basic methods available for the preparation of radiotracers Isotope exchange reactions Chemical synthesis Biochemical reactions Recoil labelling Isotope Exchange reactions: A chemical reaction in which interchange of the atoms of a given element between two or more chemical forms of the element occurs, the atoms in one form being isotopically labeled so as to distinguish them from atoms in the other form. KLEU'S College of Pharmacy 10

11. Chemical synthesis A 14 C label may be introduced into a variety of compounds by the standard synthetic procedures of organic chemistry. In addition, some new methods have been devised to conserve the radionuclides being used. When chemical synthesis is at all possible it is usually the method of choice. Synthetic method gives the greatest control over yield, position of label and purity of the product. Chemical synthesis of labelled compounds suffers from some limitations and problems such as the amount and cost of radioactive starting material. Another disadvantage of chemical synthesis is that when it is used to produce certain biologically important compounds such as amino acids, a racemic mixture results which may lead to undesirable confusion during investigation. E.g. Radiotracer synthesis from [ 11 C] iodomethane. KLEU'S College of Pharmacy 11

12. Biosynthesis/Biochemical reactions: Living organisms or active enzyme preparations, offers a biochemical means of synthesizing certain labeled compounds that are not available by chemical synthesis. This includes both the macromolecules (proteins, Polysaccharides, nucleic acids, etc.) and many simpler molecules (vitamins, hormones, amino acids and sugars). Few factors should be considered while production of labeled compound by biosynthesis First, an organism must be selected that will synthesize and accumulate practical quantities of desired compound. Culture conditions must be established so as to provide optimal yields of desired radiolabeled compounds. Isolation and purification as well as determination of distribution pattern of the label must planned accordingly. E.g. Biochemical synthesis of 11 C and 13 N. KLEU'S College of Pharmacy 12

13. Recoil labeling: In recoil labeling a recoil atom enters a given molecule. It causes a chemical change by breaking the existing chemical bonds but also give rise to new bonds for production of marked compounds. The method used recoil labelling is tritium labeling. E.g . Recoil labelling of flourine-18 labelled chlorofluoromethanes and tetrafluoromethane. Tritium Labeling: Compounds may be labeled with tritium by several methods. By reduction of unsaturated precursors The method of choice for labeling with tritium is the reduction of a suitable unsaturated precursor containing a double bond carbonyl group etc. with carrier free tritium gas or tritiated metal hydrides.       KLEU'S College of Pharmacy 13

14. By exchange reactions: Random tritium labeling may be secured by simple exchange methods, with or without catalytic action.   By g as exposure: In this method, the compound to be labelled is exposed to curie amounts of carrier free tritium gas in a sealed reaction vessel for a period of a few days to several weeks. Hence giving rise to tritium labelled compounds. KLEU'S College of Pharmacy 14

15. Application of radiotracers: Radiotracers in metabolic studies: Metabolic studies can be divided into two categories viz. primary and secondary metabolism. Primary metabolism are vital to living organism and secondary metabolites play valuable role in pharmaceuticals, food, flavors etc. E.g. The biochemical pathways of carbon in photosynthesis: Photosynthesis is a multistep process and very complex. When chlorella cells were grown in a medium containing 14 CO 2 , it was found that radioactive carbon from the carbon dioxide turned up in glucose molecules within 30 seconds after the starting of photosynthesis . For identification of steps or pathway, compounds containing 14 C were separated by chromatography and detected by autoradiography. In this way the pathway of CO 2 fixation of phosphoglyceric acid and its transformation products ending with glucose were identified. KLEU'S College of Pharmacy 15

16. ADME studies: Radiolabeled compounds are excellent investigative tools and are widely used to carry out ADME studies during drug development stages. The most commonly used radioisotopes for ADME studies are 14 C and 3 H (tritium). For in vitro studies, radiolabeled probes are utilized to test affinity with various transporters, to perform metabolism comparison among species and to assess possible formation of reactive metabolites. For in vivo studies, radiolabeled compounds are employed to identify and elucidate metabolites formed, to investigate the extent of absorption, pre systemic metabolism, bioavailability, tissue distribution and routes of excretion. Thus radioisotopes have proven to be an indispensable tool in biomedical research and have played an important role in investigation of ADME properties of new chemical entities over past few decades. KLEU'S College of Pharmacy 16

17. Tritiated Thymidine: Tritiated Thymidine was first employed in the investigation of chromosomes. Today, it is used in many different immunological tests and has become a standard for studies in cell proliferation. Tritiated Thymidine also proved useful in studies of cell migration and growth throughout the body. The few of the radiotracers which are being used for research are tritaited thymidine, 32 P nucleotides, 14 C labelled amino acids, 14 C labelled glucose, steroids, plant growth regulators and phytochemicals . Radioiodine ( 131 I ) Bioavailability of iodine in iodized salt is investigated using radioiodine (Iodine- 131). Bio-distribution using radioiodine helped to determine the bioavailability of iodine in the human body. KLEU'S College of Pharmacy 17

18. Drug Delivery: Polymers such as thermosensitive polymers are now finding applications in drug delivery system development. Radiotracers have been used for the evaluation and determination of efficiency and target evaluation of these novel drug delivery systems. These polymers based drug delivery systems can be used for radiotherapy . Radioimmunoassay: Radioimmunoassay is yet another application of the radiotracer. Since development of Radioimmunoassay (RIA), variety of immunoassays and their variants have been developed in clinical, analytical and research laboratories for the detection and quantification of molecules in very minute quantities. Apart from the precise measurement they have provided an insight into basic mechanism and revolutionized our understanding of many physiological and pathological phenomenon.   KLEU'S College of Pharmacy 18

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21. Functional Imaging: Radiotracers with nuclear imaging modalities are being used in drug research and development to measure drug pharmacokinetics and drug pharmacodynamics. Radiolabeled drugs as radiotracer can serve as research tool for early identification of problems such as poor bioavailability and non-target interactions which may lead to failure later on. A unique scientific tool called Positron Emission Tomography(PET ) plays a major role in drug discovery programme . PET imaging, can be used to demonstrate the effect of a drug through a biochemical marker of processes such as glucose metabolism or blood flow. KLEU'S College of Pharmacy 21

22. Role of Radiotracers in drug development: Radiolabeled compounds have been playing an active role as radiotracers based research tools in the better understanding of the molecular behavior of lead molecules and support every stage of drug development process. This includes all the stages basic research, identifying lead molecules, ADME studies, Tissue distribution, Optimization of formulation, Pre-clinical development. Radiotracers can also be used for functional studies of organs which can be used as therapeutic index in determination of therapeutic efficacy of the drug molecules Radiotracers techniques applied in-vivo ADME studies: The most popular in vivo application of radiolabeled compounds is for ADME (absorption, distribution, metabolism, excretion) studies, pharmacokinetics ( PK), pharmacodynamics ( PD) and mass balance studies. ADME studies in human have two purposes: (a) to evaluate the mass balance of the drug and (b) to learn about its metabolism. KLEU'S College of Pharmacy 22

23. Quantitative Whole-Body Autoradiography (QWBA): QWBA in animals and dissected human tissues has emerged as an effective radiotracers technique for the assessment of drug safety. Whole body autoradiography produces images of distribution of a radiolabeled drug over the entire animal body section. The distribution of radioactivity is imaged by exposing x-ray film on to the cut tissue sections. Radioactivity levels in individual structures are quantified by determination of level of radiation effect on the film and results are expressed as quantity of drug equivalent per unit weight of tissue. QWBA is preferred method for tissue distribution studies for regulatory filing of a new drug entity. KLEU'S College of Pharmacy 23

24. Whole Body Auto Radioluminography (WBAL) This technique is different from QWBA, as it uses phosphor imaging plate, instead of x-ray film, for the detection of radioactivity which considerably enhanced the sensitivity and reduced the exposure time in autoradiography technique. The use of imaging plate (IP) has led to WBAL as a new detection method for radiation and is attractive because it possesses high sensitivity and wide dynamic range. By radioluminography , it is now possible to observe not only the two-dimensional distribution, but also possible to quantify the radioisotope uptake in various sections of specimen. KLEU'S College of Pharmacy 24

25. Micro-dosing : Micro-dosing is relatively a new concept introduced to investigate one of the major causes for failure of drug-leads during development due to the unsatisfactory pharmacokinetic (PK) or ADME parameters. This problem can be addressed great extent by studying the behavior, PK and ADME characteristics of chemical entity at an early phase of development by micro dosing with labelled chemical entity. Micro-dosing involves the administration of drug candidate in human enough to respond in cellular levels but unlikely to produce toxic effects. The basic approach is to label a candidate drug using 14 C or 3 H and then administering the radiolabeled compound to human volunteers at levels typically about 100 times lower than the proposed therapeutic dosage based on animal studies. Thus micro-dosing allows not only evaluation of PK/ADME features, but also enables standard mass balance study in humans. When drug is present in very small amount in tissue or blood after micro dosing, its detection requires other very sensitive analytical techniques such as Positron Emission Tomography (PET) or Accelerator Mass spectrometry (AMS). 11 C is most commonly used for PET and 14 C for AMS. AMS is preferred for accurate estimation of very minute amounts of 14 C in biological samples. KLEU'S College of Pharmacy 25

26. Nuclear Imaging: One of the most important new applications of radiotracers is expected to be in the area of drug efficiency assessment by nuclear imaging. Nuclear imaging procedures such as PET (Positron Emission Tomography), SPECT (Single Photon Emission Computed Tomography) and AMS (Accelerator Mass Spectrometry) are options based on the use of specially designed radiotracers called radiopharmaceuticals that can provide criteria based status of the disease progression and functionality of the disease organ. Furthermore, PET study reveals the metabolic and physiological changes and activity in target organ which is effective quantitatively to the therapeutic effect of drug action. KLEU'S College of Pharmacy 26

27. Positron Emission Tomography ( PET) Positron Emission Tomography (PET) is a nuclear medicine, functional imaging technique that produces a three-dimensional image of functional processes in the body. The system detects pairs of gamma rays emitted indirectly by a positron-emitting radionuclide (tracer), which is introduced into the body on a biologically active molecule. Positron emission tomography (PET) is a powerful imaging technique which enables in vivo examination of brain functions. It allows non-invasive quantification of cerebral blood flow, metabolism, and receptor binding. Positron emission   Operation To conduct the scan, a short-lived radioactive tracer isotope is injected into the living subject (usually into blood circulation). There is a waiting period while the active molecule becomes concentrated in tissues of interest; then the subject is placed in the imaging scanner. The molecule most commonly used for this purpose is F-18 labeled fluorodeoxyglucose (FDG), a sugar, for which the waiting period is typically an hour. During the scan, a record of tissue concentration is made as the tracer decays. KLEU'S College of Pharmacy 27

28. Radiotracers used in PET: Radionuclides used in PET scanning are typically isotopes with short half-lives such as carbon-11 (~20 min), nitrogen-13 (~10 min), oxygen-15 (~2 min), fluorine-18 (~110 min), zirconium-89 (~78.41 hours), or rubidium-82(~1.27 min). These radionuclides are incorporated either into compounds normally used by the body such as glucose (or glucose analogues), water, or ammonia, or into molecules that bind to receptors or other sites of drug action. KLEU'S College of Pharmacy 28

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31. Single-photon emission computed tomography (SPECT) Single-photon emission computed tomography (SPECT) is a nuclear medicine tomographic imaging technique using gamma rays. It is very similar to conventional nuclear medicine planar imaging using a gamma camera. However , it is able to provide true 3D information. This information is typically presented as cross-sectional slices through the patient, but can be freely reformatted or manipulated as required. The technique requires delivery of a gamma-emitting radioisotope (a radionuclide) into the patient, normally through injection into the bloodstream. On occasion, the radioisotope is a simple soluble dissolved ion, such as a radioisotope of gallium (III) whose properties bind it to certain types of tissues . Difference between PET and SPECT: SPECT is similar to PET in its use of radioactive tracer material and detection of gamma rays. In contrast with PET, however, the tracers used in SPECT emit gamma radiation that is measured directly, whereas PET tracers emit positrons that annihilate with electrons up to a few millimeters away, causing two gamma photons to be emitted in opposite directions. KLEU'S College of Pharmacy 31

32. A PET scanner detects these emissions "coincident" in time, which provides more radiation event localization information and, thus, higher spatial resolution images than SPECT (which has about 1 cm resolution). SPECT scans, however, are significantly less expensive than PET scans, in part because they are able to use longer-lived more easily obtained radioisotopes than PET KLEU'S College of Pharmacy 32

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36. Radiotracer techniques applied in-vitro : Radiolabeled compounds are also excellent in vitro research tools for target evaluation, receptor-drug binding studies, biomarker identification, formulation evolution, drug analysis, drug delivery system evaluation etc. For in-vitro studies, radiolabeled probes are utilized to test the affinity and binding of the drugs with various transporters and receptors and to compare metabolism among species and to assess possible formation of reactive metabolites. Radio-receptor assay: Radio-receptor binding has been extensively used for identifying and characterizing a large number of enzymes and receptors for targeting in every therapeutic area. The pharmaceutical researchers routinely use this technique for identifying novel molecules which can inhibit or mimic the endogenous biochemical receptors avid molecules to cell bound receptors. KLEU'S College of Pharmacy 36

37. Patents regarding Radiotracers: OPTICAL REACTION CELL AND LIGHT OTHER PUBLICATIONS SOURCE FOR [ 18 F] FLUORIDE RADIOTRACER SYNTHESIS. (United States Patent) NOVEL COMPOSITIONS FOR RADIOTRACER LOCALIZATION OF DEEP VEIN THROMBI. (United States Patent) A PROCESS AND DEVICE FOR PRODUCING PET RADIOTRACERS. (European Patent) DISPOSABLE KIT FOR PREPARATION. (United States Patent) METHOD AND DEVICE FOR QUANTIFYING THE UPTAKE OF AT LEAST ONE RADIOTRACER IN A BODY REGION OF A PATIENT OF INTEREST TO A POSITRON EMISSION TOMOGRAPHY MEASUREMENT. ( United States Patent ) KLEU'S College of Pharmacy 37

38. Marketed Products or Marketed Imaging agents : FLORBETABEN (18F )/ NeuraCeq TM : Florbetaben , a fluorine-18 (18F)-labeled stilbene derivative trade name  NeuraCeq TM ( florbetaben F18 injection) , is a diagnostic radiopharmaceutical developed for routine clinical application to visualize ß-amyloid plaques in the brain. It is indicated for Positron Emission Tomography (PET) imaging of ß-amyloid neurotic plaque density in the brains of adult patients with cognitive impairment who are being evaluated for Alzheimer’s disease (AD) and other causes of cognitive impairment Dosage : Adult: Follow injection with an IV flush of 10 mL of 0.9% NaCl . Developer/Manufacturer : Piramal Enterprises. KLEU'S College of Pharmacy 38

39. MIBITEC (600 MBq /ml solution injectable): Most widely used SPECT cardiac imaging agent, Tetrakis (2-methoxyisobutyl iso nitrile ) copper (I) tetra fluoroborate. They are approved for myocardial exploration, localization of parathyroid issue and breast cancer diagnosis. Developer/Manufacturer : Advanced Accelerator Applications (AAA) KLEU'S College of Pharmacy 39

40. IASOflu ®: Is the brand name for Sodium Fluoride 18. This PET tracer is indicated as a bone imaging agent to define areas of altered osteogenic activity. It accumulates in the vicinity of primary and metastatic malignancy in bone. Developer/Manufacturer: Advanced Accelerator Applications (AAA ) KLEU'S College of Pharmacy 40

41. IASOcholine ®: This drug is purely for diagnostic use. 18 F-choline (FCH) is intended for Positron Emission Tomography (PET). 18 F-choline is indicated for imaging in patients undergoing oncologic diagnostic procedures describing function or diseases where enhanced choline influx of specific organs or tissues is the diagnostic target. Developer/Manufacturer: Advanced Accelerator Applications (AAA) KLEU'S College of Pharmacy 41

42. Future aspect of Radiotracers: The expenditure on the development of new drug molecules has been phenomenal and has been increasing day by day. Focused efforts are necessary to tackle the problems of the cost evaluation without compromising on efficiency and safety aspects. The use of radiolabeled compounds and radiotracer techniques has shown great promise to drug developers and researchers and the technique is in-dispensable in drug research. The unique detection sensitivity and possibility of requirement of the radiotracers in small quantity that does not disturb the biochemical equilibrium of the system under study has attracted researchers. Applying radiolabeled compound based techniques such as nuclear imaging and micro-dosing in the early stages of drug development will enable the radiotracer to further establish as a research tool in the evaluation of drug directly in human and use of radiotracer will become an integral part of drug discovery and development. KLEU'S College of Pharmacy 42

43. Conclusion : One can understand the present status and enormous potential of radiopharmaceuticals both for therapy and palliative treatment. In current scenario of evidence based medicine practice, nuclear medicine i.e. radiotracers and other radiopharmaceuticals are treated as an important modality both for diagnosis and therapy and the future of nuclear medicine as “Therapeutic era”. KLEU'S College of Pharmacy 43

44. Reference : Padmanabhan D. Applications of radiolabeled compounds. Pharma Times. 2014;46(5): 13-5. Sivaprasad N. Role of Radiotracers in Drug Development. Pharma Times. 2014;46(5): 16-7. Rajan MGR. Applications of Nuclear Imaging in Pharmacy. Pharma Times. 2014;46(5): 18-26. Isin EM, Elmore CS, Nilsson GN, Thompson RA, Weidolf L. Use of radiolabeled compounds in drug metabolism and pharmacokinetic studies. Chem. Res. Toxicol . 2011;25(3):532-42. Penner N, Xu L, Prakash C. Radiolabeled absorption, distribution, metabolism, and excretion studies in drug development: why, when and how? Chem. Res. Toxicol . 2012;25(3):513-31. Chaudhari P. Small Animal Imaging facility for Preclinical Research. Pharma Times. 2014;46(5): 27-8. Dr. Banerjee S. Radiopharmaceutical Chemistry Research in the Development of Radiopharmaceuticals. Pharm Times. 2015;47(5):25-7 KLEU'S College of Pharmacy 44

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