Radiopharmaceuticals and Methods of Radiolabeling

Radiopharmaceuticals and Methods of Radiolabeling

Design of New Radiopharmaceuticals

General Considerations
Many radiopharmaceuticals are used for various nuclear medicine tests. Some of them meet most of the requirements for the intended test and therefore need no replacement. For example, 99mTc–methylene diphosphonate (MDP) is an excellent bone imaging agent and the nuclear medicine community is fairly satisfied with this agent such that no further research and development is being pursued for replacing 99mTc-MDP with a new radiopharmaceutical.

General Considerations (cont,..)
However, there are a number of other radio pharmaceuticals that offer only minimal diagnostic value in nuclear medicine tests and thus need replacement. Continual effort is being made to improve or replace such radiopharmaceuticals.

Upon scrutiny, it is noted that the commonly used radiopharmaceuticals involve one or more of the following mechanisms of localization in a given organ: Passive diffusion: 99mTc-DTPA in brain imaging, 99mTc-DTPA aerosol and 133Xe in ventilation imaging, 111In-DTPA in cisternography. Ion exchange: uptake of 99mTc-phosphonate complexes in bone.

3. Capillary blockage: 99mTc macroaggregated albumin (MAA) particles trapped in the lung capillaries. 4. Phagocytosis: removal of 99mTc-sulfur colloid particles by the reticuloendothelial cells in the liver, spleen, and bone marrow. 5. Active transport: 131I uptake in the thyroid, 201Tl uptake in the myocardium. 6. Cell sequestration: sequestration of heat-damaged 99mTc-labeled red blood cells by the spleen.

7. Metabolism: 18F-FDG uptake in myocardial and brain tissues. 8. Receptor binding: 11C-dopamine binding to the dopamine receptors in the brain. 9. Compartmental localization: 99mTc-labeled red blood cells used in the gated blood pool study. 10. Antigen-antibody complex formation: 131I-, 111In-, and 99mTc-labeled antibody to localize tumors. 11. Chemotaxis: 111In-labeled leukocytes to localize infections.

Based on these criteria, it is conceivable to design a radiopharmaceutical to evaluate the function and/or structure of an organ of interest. Once a radiopharmaceutical is conceptually designed, a definite protocol should be developed based on the physicochemical properties of the basic ingredients to prepare the radiopharmaceutical.

The method of preparation should be simple, easy, and reproducible, and should not alter the desired property of the labeled compound. Optimum conditions of temperature, pH, ionic strength, and molar ratios should be established and maintained for maximum effcacy of the radiopharmaceutical.

Once a radiopharmaceutical is developed and successfully formulated, its clinical efficacy must be evaluated by testing it first in animals and then in humans. For use in humans, one has to have a Notice of Claimed Investigational Exemption for a New Drug (IND) from the U.S. Food and Drug Administration (FDA), which regulates the human trials of drugs very strictly. If there is any severe adverse effect in humans due to the administration of a radiopharmaceutical, then the radiopharmaceutical is discarded.

Factors Influencing the Design of New Radiopharmaceuticals
The following factors need to be considered before, during, and after the preparation of a new radiopharmaceutical.

1- Compatibility When a labeled compound is to be prepared, the first criterion to consider is whether the label can be incorporated into the molecule to be labeled. This may be assessed from a knowledge of the chemical properties of the two partners. For example, 111In ion can form coordinate covalent bonds, and DTPA is a chelating agent containing nitrogen and oxygen atoms with lone pairs of electrons that can be donated to form coordinated covalent bonds.

1- Compatibility (cont,..) Therefore, when 111In ion and DTPA are mixed under appropriate physicochemical conditions, 111In-DTPA is formed and remains stable for a long time. If, however, 111In ion is added to benzene or similar compounds, it would not label them. Iodine primarily binds to the tyrosyl group of the protein.

1- Compatibility (cont,..) Mercury radionuclides bind to the sulfhydryl group of the protein. These examples illustrate the point that only specific radionuclides label certain compounds, depending on their chemical behavior.

2- Stoichiometry In preparing a new radiopharmaceutical, one needs to know the amount of each component to be added. This is particularly important in tracer level chemistry and in 99mTc chemistry. The concentration of 99mTc in the 99mTc eluate is approximately 10-9M. Although for reduction of this trace amount of 99mTc only an equivalent amount of Sn2+is needed, 1000 to 1 million times more of the latter is added to the preparation in order to ensure complete reduction.

2- Stoichiometry (cont,..) Similarly, enough chelating agent, such as DTPA or MDP, is also added to use all the reduced 99mTc. The stoichiometric ratio of different components can be obtained by setting up the appropriate equations for the chemical reactions. An unduly high or low concentration of any one component may sometimes affect the integrity of the preparation.

3- Charge of the Molecule The charge on a radiopharmaceutical determines its solubility in various solvents. The greater the charge, the higher the solubility in aqueous solution. Nonpolar molecules tend to be more soluble in organic solvents and lipids.

4- Size of the Molecule The molecular size of a radiopharmaceutical is an important determinant in its absorption in the biologic system. Larger molecules (mol. wt. >~60, 000) are not filtered by the glomeruli in the kidney. This information should give some clue as to the range of molecular weights of the desired radiopharmaceutical that should be chosen for a given study.

5- Protein Binding Almost all drugs, radioactive or not, bind to plasma proteins to variable degrees. The primary candidate for this type of binding is albumin, although many compounds specifically bind to globulin and other proteins as well. Indium, gallium, and many metallic ions bind firmly to transferrin in plasma. Protein binding is greatly influenced by a number of factors, such as the charge on the radiopharmaceutical molecule, the pH, the nature of protein, and the concentration of anions in plasma.

5- Protein Binding (cont,..) At a lower pH, plasma proteins become more positively charged, and therefore anionic drugs bind firmly to them. The nature of a protein, particularly its content of hydroxyl, carboxyl, and amino groups and their configuration in the protein structure, determines the extent and strength of its binding to the radiopharmaceutical. Metal chelates can exchange the metal ions with proteins because of the stronger affnity of the metal for the protein. Such a process is called ‘‘transchelation’’ and leads to in vivo breakdown of the complex.

5- Protein Binding (cont,..) For example, 111In-chelates exchange 111In with transferrin to form 111In-transferrin. Protein binding a¤ects the tissue distribution and plasma clearance of a radiopharmaceutical and its uptake by the organ of interest. Therefore, one should determine the extent of protein binding of any new radiopharmaceutical before its clinical use.

5- Protein Binding (cont,..) This can be accomplished by precipitating the proteins with trichloroacetic acid from the plasma after administration of the radiopharmaceutical and then measuring the activity in the precipitate.

6- Solubility For injection, the radiopharmaceutical should be in aqueous solution at a pH compatible with blood pH (7.4). The ionic strength and osmolality of the agent should also be appropriate for blood.

6- Solubility (cont,…) In many cases, lipid solubility of a radiopharmaceutical is a determining factor in its localization in an organ; the cell membrane is primarily composed of phospholipids, and unless the radioparmaceutical is lipid soluble, it will hardly diffuse through the cell membrane. The higher the lipid solubility of a radiopharmaceutical, the greater the di¤usion through the cell membrane and hence the greater its localization in the organ.

6- Solubility (cont,…) Protein binding reduces the lipid solubility of a radiopharmaceutical. Ionized drugs are less lipid soluble, whereas nonpolar drugs are highly soluble in lipids and hence easily di¤use through cell membranes. The radiopharmaceutical 111In-oxine is highly soluble in lipid and is therefore used specifically for labeling leukocytes and platelets. Obviously, lipid solubility and protein binding of a drug play a key role in its in vivo distribution and localization.

7- Stability The stability of a labeled compound is one of the major concerns in labeling chemistry. It must be stable both in vitro and in vivo. In vivo breakdown of a radiopharmaceutical results in undesirable biodistribution of radioactivity.

7- Stability (cont,..) For example, dehalogenation of radioiodinated compounds gives free radioiodide, which raises the background activity in the clinical study. Temperature, pH, and light affect the stability of many compounds and the optimal range of these physicochemical conditions must be established for the preparation and storage of labeled compounds.

8- Biodistribution The study of the biodistribution of a radiopharmaceutical is essential in establishing its efficacy and usefulness. This includes tissue distribution, plasma clearance, urinary excretion, and fecal excretion after administration of the radiopharmaceutical.

8- Biodistribution (cont,…) In tissue distribution studies, the radiopharmaceutical is injected into animals such as mice, rats, and rabbits. The animals are then sacrificed at different time intervals, and different organs are removed. The activities in these organs are measured and compared. The tissue distribution data will tell how good the radiopharmaceutical is for imaging the organ of interest. At times, human biodistribution data are obtained by gamma camera imaging.

8- Biodistribution (cont,…) The rate of localization of a radiopharmaceutical in an organ is related to its rate of plasma clearance after administration. The plasma clearance halftime of a radiopharmaceutical is defined by the time required to reduce its initial plasma activity to one half. It can be measured by collecting serial samples of blood at deferent time intervals after injection and measuring the plasma activity. From a plot of activity versus time, one can determine the half-time for plasma clearance of the tracer.

8- Biodistribution (cont,…) Urinary and fecal excretions of a radiopharmaceutical are important inits clinical evaluation. The faster the urinary or fecal excretion, the less the radiation dose. These values can be determined by collecting the urine or feces at definite time intervals after injection and measuring the activity in the samples.

8- Biodistribution (cont,…) Toxic effects of radiopharmaceuticals must also be evaluated. These effects include damage to the tissues, physiologic dysfunction of organs, and even the death of the animal.

Methods of Radiolabeling

Methods of 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 ɤemitting radionuclides have much wider applications. The latter are particularly useful for in vivo imaging of different organs.

Methods of Radiolabeling (cont,..)
These methods and various factors affecting the labeled compounds are discussed below.

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 e¤ect, they are expected to have the same biologic and chemical properties. Examples are 125I-triiodothyronine (T3), 125I-thyroxine (T4), and 14C-, 35S-, and 3H-labeled compounds. These labeling reactions are reversible and are useful for labeling iodine-containing material with iodine radioisotopes and for labeling many compounds with tritium.

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 99mTc-labeled albumin, 99mTc-DTPA, 51Cr-labeled red blood cells, and many iodinated proteins and enzymes. In several instances, the in vivo stability of the material is uncertain and one should be cautious about any alteration in the chemical and biologic properties of the labeled compound.

Introduction of a Foreign Label (cont,…)
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. For example, 99mTc binds to DTPA, gluceptate, and other ligads by chelation.

Labeling with Bifunctional Chelating Agents
In this approach, a bifunctional chelating agent is conjugated to a macromolecule (e.g., protein, antibody) on one side and to a metal ion (e.g., Tc) by chelation on the other side. Examples of bifunctional chelating agents are DTPA, metallothionein, diamide dimercaptide (N2S2), hydrazinonicotinamide (HYNIC) and dithiosemicarbazone.

Labeling with Bifunctional Chelating Agents (cont,…)
There are two methods—the preformed 99mTc chelate method and the indirect chelator-antibody method. In the preformed 99mTc chelate method, 99mTc chelates are initially preformed using chelating agents such as diamidodithiol, cyclam, and so on, which are then used to label macromolecules by forming bonds between the chelating agent and the protein.

In contrast, in the indirect method, the bifunctional chelating agent is initially conjugated with a macromolecule, which is then allowed to react with a metal ion to form a metal-chelate-macromolecule complex. Various antibodies are labeled by the latter method. Because of the presence of the chelating agent, the biological properties of the labeled protein may be altered and must be assessed before clinical use.

Although the prelabeled chelator approach provides a purer metalchelate complex with a more definite structural information, the method involves several steps and the labeling yield often is not optimal, thus favoring the chelatorantibody approach.

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. For example, vitamin B12 is labeled with 60Co or 57Co by adding the tracer to a culture medium in which the organism Streptomyces griseus is grown. Other examples of biosynthesis include 14C-labeled carbohydrates, proteins, and fats.

Recoil Labeling Recoil 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 hence a low specific activity of the labeled product.

Recoil Labeling (cont,..)
Several tritiated compounds can be prepared in the reactor by the 6Li(n,α)3 reaction. The compound to be labeled is mixed with a lithium salt and irradiated in the reactor. Tritium produced in the above reaction labels the compound, primarily by the isotope exchange mechanism, and then the labeled compound is separated.

Excitation Labeling Excitation labeling entails the utilization of radioactive and highly reactive daughter ions produced in a nuclear decay process. During b decay or electron capture, energetic charged ions are produced that are capable of labeling various compounds of interest. Krypton-77 decays to 77Br and, if the compound to be labeled is exposed to 77Kr, then energetic 77Br ions label the compound to form the brominated compound. Similarly, various proteins have been iodinated with 123I by exposing them to 123Xe, which decaysto 123I. The yield is considerably low with this method

Important Factors in Labeling
The majority of radiopharmaceuticals used in clinical practice are relatively easy to prepare in ionic, colloidal, macroaggregated, or chelated forms, and many can be made using commercially available kits. Several factors that influence the integrity of labeled compounds should be kept in mind. These factors are described briefly below.

Efficiency of the Labeling Process A high labeling yield is always desirable, although it may not be attainable in many cases. However, 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.

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.

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. For example, proteins are denatured by heating, at pH below 2 and above 10, and by excessive iodination, and red blood cells are denatured by heating.

Isotope Effect The isotope e¤ect results in di¤erent physical (and perhaps biologic) properties due to diferences in isotope weights. For example, in tritiated compounds,H atoms are replaced by 3H atoms and the diference in mass numbers of 3H and H may alter the property of the labeled compounds. It has been found that the physiologic behavior of tritiated water is different from that of normal water in the body. The isotope effect is not as serious when the isotopes are heavier.

Carrier-Free or No-Carrier-Added (NCA) State Radiopharmaceuticals tend to be adsorbed on the inner walls of the containers if they are in a carrier-free or NCA state. Techniques have to be developed in which the labeling yield is not affected by the low concentration of the tracer in a carrier-free or NCA state.

Storage Conditions 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; for example, albumin should be stored under refrigeration. Light may also break down some labeled compounds and these should be stored 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.

Specific Activity Specific activity is defined as the activity per gram of the labeled material. In many instances, high specific activity is required in the applications of radiolabeled compounds and appropriate methods should be devised to this end. In others, high specific activity can cause more radiolysis (see below) in the labeled compound and should be avoided.

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 e¤ect 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. For example, radiations from a labeled molecule can decompose water to produce hydrogen peroxide or perhydroxyl free radical, which oxidizes another labeled molecule. To help prevent indirect radiolysis, the pH of the solvent should be neutral because more reactions of this nature can occur at alkaline or acidic pH.

Radiolysis ( cont,…) 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. In essence, radiolysis introduces a number of radiochemical impurities in the sample of labeled material and one should be cautious about these unwanted products. These factors set the guidelines for the expiration date of a radiopharmaceutical.

Purification and Analysis Radionuclide impurities are radioactive contaminants arising from the method of production of radionuclides. Fission is likely to produce more impurities than nuclear reactions in a cyclotron or reactor because fission of the heavy nuclei produces many product nuclides. Target impurities also add to the radionuclidic contaminants. The removal of radioactive contaminants can be accomplished by various chemical separation methods, usually at the radionuclide production stage.

Purification and Analysis (cont,..) Radiochemical and chemical impurities arise from incomplete labeling of compounds and can be estimated by various analytical methods such as solvent extraction, ion exchange, paper, gel, or thin-layer chromatography, and electrophoresis. Often these impurities arise after labeling from natural degradation as well as from radiolysis.

Shelf Life A labeled compound has a shelf life during which it can be used safely for its 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, any additive, the labeled molecule, the nature of emitted radiations, and the nature of the chemical bond between the radionuclide and the molecule. Usually a period of three physical 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-labeled compounds varies between 0.5 and 18 hr, the most common value being 6 hr.

Radiopharmaceuticals and Methods of Radiolabeling

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Radiopharmaceuticals and Methods of Radiolabeling

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