Loyola University Medical School, Maywood, Ill.
RADIOPHARMACOLOGY
(The state of the art) LELIO G. COLOMBETTI
Science does not always progress in a continuous flow. It is true that we are constantly gaining scientific knowledge, but it is quite common that new ideas, once they are enunciated and partially developed, lie dormant for a period of time until they are resurrected by someone who understands their usefulness, and new strides forward are made. This is precisely what has happened with Radiopharmacotogy. We can consider radiopharmacology to have been born many years ago when pharmacologists, biochemists, physicians, and other scientists started studying the biological distribution of radioactive drugs. Before radiotracers were discovered, following the pathways of a drug in a living organism was very difficult and the results, in many cases, were far from conclusive. The best that could be done in most cases was to determine the uptake of the drug by an organ, to estimate a gross general distribution, and to propose an idea of its fate. With the introduction of radiotracers in the life sciences by VON HEVESY 4 in 1923, a new approach was made available to scientists, in which labeling drugs with radioactive atoms permitted one to follow closely the pathways of the drugs in vivo. Even so, no one thought of this application as the beginning of a new scientific discipline, in part because of the primitive quality of these techniques and partly because scientists working with radiolabeled drugs came from such diverse disciplines (organic chemistry, biochemistry, pharmacology, physiology, etc.) that it was difficult to organize their efforts into a new branch of science. In addition, it was difficult to obtain the needed radionudides. This problem was solved when Fr~d6ric and Ir6ne Joliot-Curie discovered how to produce radioactivity artifidally by
Key-words: Biomedical research; Drug action; Pharmacology; Radiopharmacology; Radiotracers. Accepted for publication on May 7, 1979. La Ricerca Clin. Lab. 9, 281, 1979. 281
RADIOPHARIVIACOLOGY
bombarding aluminum atoms with c~ particles in 1934 2. After this discovery, many scientists dedicated themselves to the production of new radionuclides, and in a short period of time we had in our laboratories what was needed to produce radiolabeled drugs. In spite of these strides forward, it was about fifteen years before a radioactive drug was used by a pharmacologist. In 1948 GUILING et al. 3 working at the University of Chicago, synthesized, using biological techniques, the first radio-drug: digitoxin labeled with carbon-14. Also at the University of Chicago, a few months later, ROTH et al. 10 studied the metabolism of 14C-nicotinic acid and 14C-nicotinamide in the mouse. Pharmacologists all over the world took up these ideas and soon started studying the distribution of drugs labeled with tritium and carbon-14 in animals and also in humans 9. After the discovery of the whole-body autoradiography by ULLBERCn in 1954 this technique became popular among pharmacologists for studying the deposition of drugs in animal tissues. Doing whole-body autoradiography of sulfur-35 labeled penicillin, Ullberg was able to obtain an immediate picture of the distribution of the radiotracer in the body of a mouse. Modern techniques such as the use of computers 8 were later introduced to facilitate quantitative studies. Even after these advances, no cohesive scientific framework had yet been formed to back-up the work of these pioneers, and each scientist was on his own trying to discover the biological pathways that account for the disposition of chemicals. About 20 years ago, when the first radiolabeled drugs were introduced for application in diagnostic medicine, it was understood by a number of researchers that a new scientific field was being opened for them. The beginnings of this new field were very modest. Most of the scientists involved were chemists and biochemists with little or no training in pharmacology, or pharmacologists and physicians with insufficient training in chemistry. It was a great thrill for a chemist to perform an autopsy on a rat or mouse, or for a pharmacologist to try to label a drug with a radionuclide. But the radiotracers were needed and needed immediately, and so many of us had to improvise laboriously experimenting in areas unfamiliar to us and suffering many failures. I still remember the time, about 20 years ago, when I performed my first autopsy on a mouse to study the distribution of my first labeled drugs, by counting aliquots of the organs involved. Rather than dissecting the poor creature, I butchered it. But the information was needed, the task had to be done, and we did it! I believe that at that time there were very few scientists with the specialized training to do a complete job, but we felt great pressure from the medical community to fulfill the expectations created by those pioneering physicians in diagnostic nuclear medicine. So we channeled all our efforts toward meeting this challenge. Time was very short because this new medical technique was very expensive and those pioneers had to prove its usefulness practically overnight, so that support for programs would not be withdrawn by the government or the hospital administrators. At that time the clinical information gained was far more important than knowing the cause or reasons for the distribution of a drug or its specific localization. So little time was available for these studies, that during the first five to ten years of our work, very few scientists attempted thorough study of the pharmacokinetics of the new radiolabeled drugs. However, as some individuals started looking for 282
L.G. COLOMBETTI
the reasons behind specific or preferential distribution of these chemicals and the number of scientists working in the field became greater, more and more of the basic work was done. CONTRIBUTIONS TO BIOMEDICAL RESEARCH
Probably one of the most important contributions of radiotracers to biology is the proof that all biological units in a living organism are in a state of continuous change. This continuous change can be demonstrated by using radiotracers, whose dynamics, mechanisms of transport and localization, catabolic pathways, and other parameters can be quantitatively measured. Since classical chemical and biological methods are limited to measurements of intake and excretion or to direct observations of concentrations in accessible body compartments, these methods can yield little information about the dynamic nature of a system. Only by altering the steady state of a system can the dynamics be studied by these techniques; such alterations, however, introduce undesirable extraneous effects. Radiotracers, on the other hand, do not disturb the equilibrium of the system, yet are not themselves in a steady state when introduced, so that their transport, localization, and metabolic fate can be analyzed as a function of time. The number and types of applications of radiotracers in biology and medicine have been increasing very rapidly during the last few years. Today, it is possible to study very complicated biological processes such as enzyme induction or the delay in urinary excretion caused by a defect in the renal filtration mechanism. It is possible to study the stimulating or depressing effects of drugs on the glands of the endocrine system. Based on preferential localization of radiotracers, we can study the extent of infarcts. Blood perfusion of organs is not a mystery anymore. Using labeled chemicals, one is able to localize the point of attachment of a chemical within the cell even ff the chemical does not have a pharmacological action. Many other problems involved with the physiology of an organism can be pinpointed, facilitating diagnosis and prognosis in a clinical setting. WHAT IS 'RADIOPHARMACOLOGY'?
As is often the case when a new idea is in the early stage of development, it is not easy to make a concise and precise statement of what we 'really' mean when we talk about radiopharmacology. In spite of the fact that a definition could be more confusing than edifying at the present time, I will try to explain the essential meaning of this term, and only then venture a definition. Pharmacology, as a science, started about 200 years ago, when Felix Fontana advanced the idea that each active component of a crude drug exerts its own characteristic effect at one or more specific sites of the body 6. It was possible for Magendle to confirm this idea experimentally fifty years later, when the chemists could provide pure drugs and the physiologists, the experimental methods. Because scientists working in this area could now identify and organize the materials and technique needed to advance this discipline in its own right, we can consider that at this time the 'pharmacologist' was born. From this time on, no great lapses occurred between significant findings: new developments in all aspects of pharmacology followed one another in rapid succession. To make a long story short, the 283
RADIOPHARMACOLOGY
most important contribution to this development, for our purposes, proved to be the application of labeled drugs to the study of the mechanisms of drug distribution and action ~. About four years ago, McArEE 7 in an analysis of the future of radiopharmaceuticals in medicine, stated that the "radiopharmaceutical field can now progress beyond the stage of mere radiopharmacy to radiopharmacology" confirming our thoughts of a few years earlier that the teaching of such a branch of science was becoming absolutely necessary. But this idea was not new at the time when McAfee made this statement: five years earlier, Louis Bugnard of the University of Paris was already asking that the scientific community "have regular meetings of groups of workers interested in the problems of radiopharmacology" 5. Did Bugnard coin the word 'radiopharmacology'? I do not know, but this is the first mention of it I could find in the literature. McAfee continues by saying that "this can be accomplished by making full use of the knowledge gained about the biological disposition of drugs and using a more rational approach by understanding the mechanisms of localization of drugs" 7 A definition of radiopharmacology could be as-difficult to understand as what we mean when we talk about radiopharmaceuticals. McAFEE 7 has clearly stated that the term radiopharmaceuticals is a misnomer, since it denotes diagnostic agents that have no significant pharmacological effects at the doses ordinarily used. However, radiotracers applied to medicine are universally known as radiopharmaceuticals. I suppose it is so because of lack of a better term.., and I believe that the same reasoning may have to suffice fo r radiopharmacology. As stated by Schmiedeberg over a century ago 6 "the purpose of pharmacology is to study the reactions brought about in living organisms by chemical substances". Since the use of the common small doses of radiotracers does not result in a measurable pharmacological action ~, they do not fall within the strict scope of pharmacology. But I believe that a radiotracer will behave in a living organism in the same manner as a non-radioactive drug: their mechanisms of transport and localization and the fate of the metabolites will be the same. Therefore, by association, we can say that radiopharmacology is the science that studies the chemical properties of radiotracers (or radiopharmaceuticals) and their interactions with living organisms. It is evident that, so defined, radiopharmacology is an interdisciplinary science that, within the context of medicine, integrates the appropriate scientific and technological components of the broad, traditional fields of chemistry, physics, and biology, particularly the recognized specialities of biochemistry and nuclear chemistry (which are themselves interdisciplinary areas), physiology and nuclear physics. Schmiedeberg, who can be considered the first professor of pharmacology, created a teaching program at Strasbourg University, and also founded the first journal devoted to reports of pharmacologic experimentation, the Archiv ]iir experimentelle Pathologic und Pharmakologie. Like him, about seven years ago, we developed a teaching program for those interested in the new science of radiopharmacology. T H E T E A C H I N G OF RADIOPHARMACOLOGY
About 10 years ago, Marshall BRUCER1 stated that Rutherford created nuclear physics and Becquerel, nuclear chemistry but, of course, they did not invent those 284
L, G. COLOMBETTI
names. At that time nuclear science was so poorly defined that Becquerel, a chemist, won his Nobel Prize in 1903 for physics while Rutherford, a physicist, won his in chemistry in 1908. With the new developments in these areas of science, we started understanding better what the differences between nuclear chemistry and nuclear physics are or, better yet, what they are not. The separation of these two scientific disciplines is almost impossible, just as it is difficult to distinguish radiopharmacology from nuclear chemistry, nuclear physics, biochemistry, etc... Why? Because a bit of each areas of science put together make radiopharmacology. Because of the diversity in this field, one cannot expect good radiopharmacologists to appear spontaneously from the established pedagogic pathways - such as physiology or nuclear physics - that emphasize certain parts of the background needed for radiopharmacology while disregarding others. Fully qualified practitioners of radiopharmacology can best be produced by a program that is deliberately oriented toward its specific objective; such a program represents a conscious synthesis of the courses for teaching all of the required intellectual and practical skills, either by adopting those courses from existing curricula or b y creating them 'ab initio'. About seven years ago, we realized that the time had come to develop a program to prepare scientists working in the field. Learning, as is well known, is an active process - a student cannot simply pay passive attention to textbooks, lectures and demonstrations. It is necessary for the student to use what he or she hears, sees, and does, applying the newly acquired knowledge in reasoning out the kinds of problems that will be encountered in the future. For example, in mastering the basic principles of radiopharmacology, each student must become capable of deducing the possible biological distribution of a radioactive tracer in order to understand its actions, side effects, usefulness, and other characteristics. I, like many of my friends, have learned from personal experience how difficult, slow, and unreliable it is to improvise techniques and equipment and to proceed by trfal and error in a field of endeavour for which no methodology has been laid down by earlier workers. These efforts, though sometimes highly rewarding, tend to be haphazard and inefficient, and they cannot be recommended as a preferred pattern for training the next generation of radiopharmacologists. In the past, drugs have been found and developed through different approaches. At the very beginning, chance observation of the actions of natural products led to deliberate use of the substances with therapeutic effects. Then, as medicine developed into a science, a systematic approach was adopted that accelerated the discovery of new drugs. Today, many thousands of drugs are known. The conversion of a few of these into radioactive tracers has been a great achievement by the scientists working in this field. These initial advances have been made on the basis of chance, empirical methods of formulation, and animal testing. It is evident that such an incompletely organized approach can lead to the development of only small numbers of sensitive and specific radiotracers. Because facts, theories, and ideas are retained only to the extent that they are used, we emphasize in our program the practical applications of the theories Iearned. Laboratory animal work, as well as chemical experimentation, is of prime importance in our program, and the students are given as many opportunities as possible to do the work themselves. The main purpose of our program is to prepare scientists capable of developing new radiotracers with known, specific localizations and 285
RADIOPHARMACOLOGY
biological pathways and new radioactive drugs to be used in medicine for diagnostic or therapeutic ends. To make a rational prediction of the localization and biological behaviour of a tracer, a basic general scientific background is necessary. Several branches of knowledge, including chemistry, biochemistry, physiology, nuclear chemistry and physics, and pharmacokinetics, should be thoroughly mastered before the design of a new radiotracer is attempted. The effects of solute structures, binding proteins, and other components of the blood upon the radiotracer should be clearly understood. The appropriate approaches to the design of radiotracers must be based on informed comprehension of the influences of chemical structure, solubility, molecular or particle size, stability in vivo, as well as in vitro, and many other variables related to the application of radiotracers in humans. The problem, therefore, is the preparation of scientists having these combined skills. The lack of a program to prepare scientists to perform this task has stimulated us to create a course in radiopharmacology. The course is intended to cover, in a comparative, didactic, and practical fashion, those fundamental and clinical sciences that are the basis for the useful application of radiotracers in medicine. Lectures, laboratory exercises, and demonstrations form the core of the program, and opportunities to solve practical problems are provided to each individual. I believe this to be a pioneer program, unique in its conception and scope. This program provides graduate training in basic and applied radiopharmacology in order to render its participants capable of designing new radiotracers and understanding all the mechanisms involved in the use of radiotracers in biological research and in nuclear medicine. Candidates for this curriculum which leads to a Ph.D. degree in radiopharmacology, must have at least a master's degree in physics, biochemistry, or chemistry or a degree in medicine or pharmacology. The program is divided into two basic parts. The first part comprises a review of scientific knowledge applicable to the field of radiopharmacology. Depending on their previous training, the candidates take those courses necessary to complete the needed scientific background before entering the second part of the program. The basic courses that are mandatory in the first part are physics, chemistry (inorganic, organic, synthesis, etc.), anatomy and physiology, biochemistry, pharmacology, and toxicology. Mastery of these topics ensures that the basic knowledge and working tools are understood by everybody taking the second part of the program, in which the application of radiotracers in biology is taught. The length of the first part of the program will depend on the academic background of the candidate, and the courses can be taught as standard courses in different schools of the University. The second part of the program requires two semesters and is divided into five areas, each represented by several courses; 40% of the time is spent in lectures, conferences, and group demonstrations. The other 60% involves laboratory work in which mathematical problems have to be solved; different compounds have to be labeled with various radionuclides, including those of carbon, hydrogen, iodine, chromium, technetium, and other elements; animal experimentation must be mastered, including minor and major surgery in rodents, cats, and dogs; and proficiency must be acquired in tissue preparation for autoradiography and scintillation counting. The courses that make up the second part of the program are the following: 286
L.G. COLOMBETTI
A.
BASIC TECHNOLOGY
1. Mathematics algebra: exponentials; logarithms; proportions; analytic geometry; differential calculus; integral calculus; differential equations; dimensional analysis; probability and statistics. 2. Nuclear physics atomic and nuclear theory; contemporary atomic and nuclear physics; the radioactive nucleus: a, ~ and y decays; radioactive decay series; parent and daughter nuclides; production of radionuclides; acceleration of charged particles; detection of radioactivity; radionuclides for medical applications; dosimetry; radiation protection. 3. Nuclear chemistry chemical foundations of atomic theory; nuclear reactions: classification, production, and study; interaction of radiation with matter; biological effects of radiation; tracers in chemical applications; the radiotracer laboratory; radiochemical separations. B. RADIOTRACERS
1. Radiotracer design introduction; mathematical models; classical design concepts: pharmacological and physiological bases; design of binding sites. . Preparation concepts and criteria; tracer applications; radiolabeling, isotopic and non-isotopic; synthesis of 14Cradiotracers; biosynthetic procedures. . Chemical properties radionuclidic purity; radiochemical purity; carrier-free radiotracers; stability of radiotracers: chemical effects, radiolysis. 287
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4. Biological properties radiotracer receptors: molecular properties; radiotracer localization: ligand-receptor interactions; radiotracer interaction with hormone receptors, enzyme receptors, etc.; metabolism of radiotracers: stimulation and depression demonstration in vitro biological fate of radiotracers biological experimentation with radiotracers liquid scintillation and ~, counting, including tissue preparation autoradiography, including tissue preparation side effects of radiotracers data analysis in radiotracer studies: applications of computers absorbed radiation dose in radiotracer studies. 5. Biological transport general considerations; membranes: composition, structure, and function; transport of radiotracers: kinetics and thermodynamics effects of solute structure and binding proteins distribution of radioions and radiolabeled metabolites; techniques of studying the transport of radiotracers. 6. Mechanisms of localization a. Compartmental localization: theory and general considerations passive transfer: distribution of dilution localization by diffusion specialized transfer: active transport. b. Radiotracer study oJ cellular function individual organs or systems; brain: 14C-glucose; muscle: myocardium imaging; kidney: secretion and filtration; endocrine system: thyroid: trapping and metabolism of iodines other endocrine functions liver: hepatocyte function microsomal function: measurement in vivo skeletal system: radioion exchange in bone cells bone physiology demonstrated by radiotracer kinetics respiratory system: radioactive gases circulatory system: trapping and clearance of macroparticles clot formation and disposition of radiotracers.
288
L.G. COLOMBETTI
c. General processes o] radiotracer localization localization of free radioions in tumour cells; cell trapping of amino acids; phagocytosis and pinocytosis; other localizations observable in vivo; metabolism of radiotracers: demonstration in vitro; radiobioassays in demonstrating physiological changes: competitive protein-binding radioimmunoassay, etc.
THE FUTURE
OF RADIOPHARMACOLOGY
I believe that in establishing radiopharmacology as a new branch o f science, the first, most important steps have been taken. When the present efforts to organize scientists involved in this area are successful, I think that radiopharmacology will be able to stand by itself in the future. In order to spread the information gained by us, so others may take advantage of what has already been done and move toward the full integration of radiopharmacology into the biological sciences, very important steps have been taken. The first step has been the organization of the teaching program, that I described in the preceding pages; this program has proved invaluable in the preparation of scientists with the necessary skills to work in the development and applications of radiotracers. The second effort has been the organization of the First International Symposium on Radiopharmacology, which was held in Innsbruck, Austria, in May 1978. This symposium, sponsored by many important organizations, was extraordinarily successful. More than 380 scientists from 37 countries came to Innsbruck to participate in the symposium. The symposium concentrated mainly on an education program based on our teaching experience, and in which the fundamental aspects and biological applications of radiotracer technology were discussed. The symposium focused on the design and preparation of radiotracers, the interactions of radiotracers with binding sites, mechanisms of transport and localization of radiotracers, uptake and binding of radiotracers in the cells, fate of radiometabolites, and medical applications. Communications bearing on the transport and metabolism of radiotracers and the fate of radiometabolites were also presented. The interest shown by scientists in this symposium guarantees the success of the second international symposium, for which a site in the USA was chosen in a meeting of delegates from 34 countries during the Innsbruck symposium. The most recent step taken up to this time has been the organization of the International Association of Radiopharmacology, which has been functioning with temporary headquarters in Chicago. The association was founded after a worldwide poll was taken in which questionnaires were sent to more than 2,000 scientists from 48 countries. Results of the poll indicated almost 100% support for a second international symposium on radiopharmacology (only two responses were negative); 83.6% were in favour of creating the association. The main purpose of this association is to bring together all those interested in the uses of radiotracers for biological research and to organize the international and national meetings dedicated exclusively to this branch of science. The first central committee of the association includes the following members: Dr. Lelio G. Colombetti (Chicago), President; 289
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Dr. Kinichi Hisada (Kanazawa, Japan), Vice-President; Dr. Howard Glenn (Houston), Secretary; and Dr. W. Earl Barnes (Chicago), Treasurer. This committee is preparing a draft of the constitution and bylaws, which will be presented to all those who showed interest in the association. All scientists working with radiotracers for biological, including medical, applications are invited to become members of the association and join us in the development and uses of radiotracers for medical applications. W e believe that together we can make giant strides forward in developing all the tracers needed in medicine and in solving many biological problems that are today difficult to comprehend.
CONCLUSIONS "~Te attempted to present an objective view of the development of radiopharmacology and the importance of adapting an organized approach to the study of radiotracers in medical science. The use of radiotracers in diagnostic and therapeutics has been steadily increasing. Radiotracers have provided the medical science with a new methodology for studying the mechanisms of transport and action of drugs, have made possible the study of metabolism, and have helped tremendously in the prognosis of disease. ~Te foresee that teaching programs will be created in which scientists and physicians will be trained as researchers in this new area of science to fulfill the present and future needs.
SUMMARY The past, present and future development of radiopharmacology as a new branch of science is being presented. Radiopharmacological techniques have been applied by many scientists in the past, not knowing that they were developing a new scientific field. With the advancement of these techniques, the creation of a specialized teaching program and the organization of the first international symposium on radiopharmaeology, the first specialized meeting in this area, radiopharmacology became a discipline standing on its own feet. The main purpose of radiopharmacology is to study the chemical properties of radiotracers, and their interactions with living organisms. In order to promote and further expand this field, an international association of radiopharmacology was created. The main purpose of the association is to congregate all those interested in the uses of radiotracers in biological sciences, including medicine, by the organization of national and international meetings. REFERENCES 1. BRUCERM.A.: A tracer has no pharmacology - In: Vignettes in nuclear medicine. MaUinckrodt Nuclear Publ., St. Louis, Mo., 1966; chapt. 24. 2. CURm I., JOLIOXF.: Artificial production of a new kind of radioelement - Nature (Lond.) 153, 201, 1934. 3. GEXLXNGE. M. K., KELSEYF.E., MCINTOSHB.J., GANZA.: Biosynthesis of radioactive drugs using carbon-14 - Science I08, 588, 1948. 4. HEV~.SYVONG.: The absorption and translocafion of lead by plants - Biochem. J. I7, 439, 1923. 5. INTERNATIONALEncyclopedia of Pharmacology and Therapeutics (IUPHAR): COHENY. (Ed.): Pergamon Press, New York, 1971; 1st ed., forward section 78, vol. 1. 6. LEVmE R.R.: Pharmacology: drug actions and reactions - Little, Brown and Co., Boston, 1978; 2nd ed., p. 8. 7. McAFEE J.G.: Radioactive diagnostic agents: current problems and limitations - In: SUBRAMAmAN G., RHODES B.A.,. COOVER J.F., SODD V.J. (Eds): Radiopharmaceuticals. Society of Nuclear Medicine, New York, 1975; p. 3. 290
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8. NODINEJ.H., PLATT J.M., CARRANZAJ., DYKYJ R., MAPP Y.: Digital computer analysis of human isotopic drug kinetics - Int. J. appl. Radiat. 15, 263, 1964. 9. OKITA G.T., TALSO P.J., CURRY J.H., SMITH F.D., GEILING E.M.K.: Metabolic fate of radioactive digitoxin in human subjects - J. Pharmacol. exp. Ther. I15, 371, 1955. 10. ROTH L.J., LErER E., HOGNESS J.R., LANGHAMW. FI.: Studies of the metabolism of radioactive nicotinic acid and nicotinamide in mice - J. biol. Chem. t76, 249, 1948. 11. ULLBERG S.: Studies of the distribution of 35Sdabelled benzylpeniciUin in the body - Acta radiol. (Stockh.) 1IS(Suppl.), 1, 1954.
Requests/or reprints should be addressed to: LELIO G. COLOMBETTI Michael Reese Hospital and Medical Center Division o/ Nuclear Medicine 29th Street and Ellis Avenue Chicago, Ill. 60616 - USA
291
RADIOPHARMACOLOGY
(The state of the art) LELIO G. COLOMBETTI
Science does not always progress in a continuous flow. It is true that we are constantly gaining scientific knowledge, but it is quite common that new ideas, once they are enunciated and partially developed, lie dormant for a period of time until they are resurrected by someone who understands their usefulness, and new strides forward are made. This is precisely what has happened with Radiopharmacotogy. We can consider radiopharmacology to have been born many years ago when pharmacologists, biochemists, physicians, and other scientists started studying the biological distribution of radioactive drugs. Before radiotracers were discovered, following the pathways of a drug in a living organism was very difficult and the results, in many cases, were far from conclusive. The best that could be done in most cases was to determine the uptake of the drug by an organ, to estimate a gross general distribution, and to propose an idea of its fate. With the introduction of radiotracers in the life sciences by VON HEVESY 4 in 1923, a new approach was made available to scientists, in which labeling drugs with radioactive atoms permitted one to follow closely the pathways of the drugs in vivo. Even so, no one thought of this application as the beginning of a new scientific discipline, in part because of the primitive quality of these techniques and partly because scientists working with radiolabeled drugs came from such diverse disciplines (organic chemistry, biochemistry, pharmacology, physiology, etc.) that it was difficult to organize their efforts into a new branch of science. In addition, it was difficult to obtain the needed radionudides. This problem was solved when Fr~d6ric and Ir6ne Joliot-Curie discovered how to produce radioactivity artifidally by
Key-words: Biomedical research; Drug action; Pharmacology; Radiopharmacology; Radiotracers. Accepted for publication on May 7, 1979. La Ricerca Clin. Lab. 9, 281, 1979. 281
RADIOPHARIVIACOLOGY
bombarding aluminum atoms with c~ particles in 1934 2. After this discovery, many scientists dedicated themselves to the production of new radionuclides, and in a short period of time we had in our laboratories what was needed to produce radiolabeled drugs. In spite of these strides forward, it was about fifteen years before a radioactive drug was used by a pharmacologist. In 1948 GUILING et al. 3 working at the University of Chicago, synthesized, using biological techniques, the first radio-drug: digitoxin labeled with carbon-14. Also at the University of Chicago, a few months later, ROTH et al. 10 studied the metabolism of 14C-nicotinic acid and 14C-nicotinamide in the mouse. Pharmacologists all over the world took up these ideas and soon started studying the distribution of drugs labeled with tritium and carbon-14 in animals and also in humans 9. After the discovery of the whole-body autoradiography by ULLBERCn in 1954 this technique became popular among pharmacologists for studying the deposition of drugs in animal tissues. Doing whole-body autoradiography of sulfur-35 labeled penicillin, Ullberg was able to obtain an immediate picture of the distribution of the radiotracer in the body of a mouse. Modern techniques such as the use of computers 8 were later introduced to facilitate quantitative studies. Even after these advances, no cohesive scientific framework had yet been formed to back-up the work of these pioneers, and each scientist was on his own trying to discover the biological pathways that account for the disposition of chemicals. About 20 years ago, when the first radiolabeled drugs were introduced for application in diagnostic medicine, it was understood by a number of researchers that a new scientific field was being opened for them. The beginnings of this new field were very modest. Most of the scientists involved were chemists and biochemists with little or no training in pharmacology, or pharmacologists and physicians with insufficient training in chemistry. It was a great thrill for a chemist to perform an autopsy on a rat or mouse, or for a pharmacologist to try to label a drug with a radionuclide. But the radiotracers were needed and needed immediately, and so many of us had to improvise laboriously experimenting in areas unfamiliar to us and suffering many failures. I still remember the time, about 20 years ago, when I performed my first autopsy on a mouse to study the distribution of my first labeled drugs, by counting aliquots of the organs involved. Rather than dissecting the poor creature, I butchered it. But the information was needed, the task had to be done, and we did it! I believe that at that time there were very few scientists with the specialized training to do a complete job, but we felt great pressure from the medical community to fulfill the expectations created by those pioneering physicians in diagnostic nuclear medicine. So we channeled all our efforts toward meeting this challenge. Time was very short because this new medical technique was very expensive and those pioneers had to prove its usefulness practically overnight, so that support for programs would not be withdrawn by the government or the hospital administrators. At that time the clinical information gained was far more important than knowing the cause or reasons for the distribution of a drug or its specific localization. So little time was available for these studies, that during the first five to ten years of our work, very few scientists attempted thorough study of the pharmacokinetics of the new radiolabeled drugs. However, as some individuals started looking for 282
L.G. COLOMBETTI
the reasons behind specific or preferential distribution of these chemicals and the number of scientists working in the field became greater, more and more of the basic work was done. CONTRIBUTIONS TO BIOMEDICAL RESEARCH
Probably one of the most important contributions of radiotracers to biology is the proof that all biological units in a living organism are in a state of continuous change. This continuous change can be demonstrated by using radiotracers, whose dynamics, mechanisms of transport and localization, catabolic pathways, and other parameters can be quantitatively measured. Since classical chemical and biological methods are limited to measurements of intake and excretion or to direct observations of concentrations in accessible body compartments, these methods can yield little information about the dynamic nature of a system. Only by altering the steady state of a system can the dynamics be studied by these techniques; such alterations, however, introduce undesirable extraneous effects. Radiotracers, on the other hand, do not disturb the equilibrium of the system, yet are not themselves in a steady state when introduced, so that their transport, localization, and metabolic fate can be analyzed as a function of time. The number and types of applications of radiotracers in biology and medicine have been increasing very rapidly during the last few years. Today, it is possible to study very complicated biological processes such as enzyme induction or the delay in urinary excretion caused by a defect in the renal filtration mechanism. It is possible to study the stimulating or depressing effects of drugs on the glands of the endocrine system. Based on preferential localization of radiotracers, we can study the extent of infarcts. Blood perfusion of organs is not a mystery anymore. Using labeled chemicals, one is able to localize the point of attachment of a chemical within the cell even ff the chemical does not have a pharmacological action. Many other problems involved with the physiology of an organism can be pinpointed, facilitating diagnosis and prognosis in a clinical setting. WHAT IS 'RADIOPHARMACOLOGY'?
As is often the case when a new idea is in the early stage of development, it is not easy to make a concise and precise statement of what we 'really' mean when we talk about radiopharmacology. In spite of the fact that a definition could be more confusing than edifying at the present time, I will try to explain the essential meaning of this term, and only then venture a definition. Pharmacology, as a science, started about 200 years ago, when Felix Fontana advanced the idea that each active component of a crude drug exerts its own characteristic effect at one or more specific sites of the body 6. It was possible for Magendle to confirm this idea experimentally fifty years later, when the chemists could provide pure drugs and the physiologists, the experimental methods. Because scientists working in this area could now identify and organize the materials and technique needed to advance this discipline in its own right, we can consider that at this time the 'pharmacologist' was born. From this time on, no great lapses occurred between significant findings: new developments in all aspects of pharmacology followed one another in rapid succession. To make a long story short, the 283
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most important contribution to this development, for our purposes, proved to be the application of labeled drugs to the study of the mechanisms of drug distribution and action ~. About four years ago, McArEE 7 in an analysis of the future of radiopharmaceuticals in medicine, stated that the "radiopharmaceutical field can now progress beyond the stage of mere radiopharmacy to radiopharmacology" confirming our thoughts of a few years earlier that the teaching of such a branch of science was becoming absolutely necessary. But this idea was not new at the time when McAfee made this statement: five years earlier, Louis Bugnard of the University of Paris was already asking that the scientific community "have regular meetings of groups of workers interested in the problems of radiopharmacology" 5. Did Bugnard coin the word 'radiopharmacology'? I do not know, but this is the first mention of it I could find in the literature. McAfee continues by saying that "this can be accomplished by making full use of the knowledge gained about the biological disposition of drugs and using a more rational approach by understanding the mechanisms of localization of drugs" 7 A definition of radiopharmacology could be as-difficult to understand as what we mean when we talk about radiopharmaceuticals. McAFEE 7 has clearly stated that the term radiopharmaceuticals is a misnomer, since it denotes diagnostic agents that have no significant pharmacological effects at the doses ordinarily used. However, radiotracers applied to medicine are universally known as radiopharmaceuticals. I suppose it is so because of lack of a better term.., and I believe that the same reasoning may have to suffice fo r radiopharmacology. As stated by Schmiedeberg over a century ago 6 "the purpose of pharmacology is to study the reactions brought about in living organisms by chemical substances". Since the use of the common small doses of radiotracers does not result in a measurable pharmacological action ~, they do not fall within the strict scope of pharmacology. But I believe that a radiotracer will behave in a living organism in the same manner as a non-radioactive drug: their mechanisms of transport and localization and the fate of the metabolites will be the same. Therefore, by association, we can say that radiopharmacology is the science that studies the chemical properties of radiotracers (or radiopharmaceuticals) and their interactions with living organisms. It is evident that, so defined, radiopharmacology is an interdisciplinary science that, within the context of medicine, integrates the appropriate scientific and technological components of the broad, traditional fields of chemistry, physics, and biology, particularly the recognized specialities of biochemistry and nuclear chemistry (which are themselves interdisciplinary areas), physiology and nuclear physics. Schmiedeberg, who can be considered the first professor of pharmacology, created a teaching program at Strasbourg University, and also founded the first journal devoted to reports of pharmacologic experimentation, the Archiv ]iir experimentelle Pathologic und Pharmakologie. Like him, about seven years ago, we developed a teaching program for those interested in the new science of radiopharmacology. T H E T E A C H I N G OF RADIOPHARMACOLOGY
About 10 years ago, Marshall BRUCER1 stated that Rutherford created nuclear physics and Becquerel, nuclear chemistry but, of course, they did not invent those 284
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names. At that time nuclear science was so poorly defined that Becquerel, a chemist, won his Nobel Prize in 1903 for physics while Rutherford, a physicist, won his in chemistry in 1908. With the new developments in these areas of science, we started understanding better what the differences between nuclear chemistry and nuclear physics are or, better yet, what they are not. The separation of these two scientific disciplines is almost impossible, just as it is difficult to distinguish radiopharmacology from nuclear chemistry, nuclear physics, biochemistry, etc... Why? Because a bit of each areas of science put together make radiopharmacology. Because of the diversity in this field, one cannot expect good radiopharmacologists to appear spontaneously from the established pedagogic pathways - such as physiology or nuclear physics - that emphasize certain parts of the background needed for radiopharmacology while disregarding others. Fully qualified practitioners of radiopharmacology can best be produced by a program that is deliberately oriented toward its specific objective; such a program represents a conscious synthesis of the courses for teaching all of the required intellectual and practical skills, either by adopting those courses from existing curricula or b y creating them 'ab initio'. About seven years ago, we realized that the time had come to develop a program to prepare scientists working in the field. Learning, as is well known, is an active process - a student cannot simply pay passive attention to textbooks, lectures and demonstrations. It is necessary for the student to use what he or she hears, sees, and does, applying the newly acquired knowledge in reasoning out the kinds of problems that will be encountered in the future. For example, in mastering the basic principles of radiopharmacology, each student must become capable of deducing the possible biological distribution of a radioactive tracer in order to understand its actions, side effects, usefulness, and other characteristics. I, like many of my friends, have learned from personal experience how difficult, slow, and unreliable it is to improvise techniques and equipment and to proceed by trfal and error in a field of endeavour for which no methodology has been laid down by earlier workers. These efforts, though sometimes highly rewarding, tend to be haphazard and inefficient, and they cannot be recommended as a preferred pattern for training the next generation of radiopharmacologists. In the past, drugs have been found and developed through different approaches. At the very beginning, chance observation of the actions of natural products led to deliberate use of the substances with therapeutic effects. Then, as medicine developed into a science, a systematic approach was adopted that accelerated the discovery of new drugs. Today, many thousands of drugs are known. The conversion of a few of these into radioactive tracers has been a great achievement by the scientists working in this field. These initial advances have been made on the basis of chance, empirical methods of formulation, and animal testing. It is evident that such an incompletely organized approach can lead to the development of only small numbers of sensitive and specific radiotracers. Because facts, theories, and ideas are retained only to the extent that they are used, we emphasize in our program the practical applications of the theories Iearned. Laboratory animal work, as well as chemical experimentation, is of prime importance in our program, and the students are given as many opportunities as possible to do the work themselves. The main purpose of our program is to prepare scientists capable of developing new radiotracers with known, specific localizations and 285
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biological pathways and new radioactive drugs to be used in medicine for diagnostic or therapeutic ends. To make a rational prediction of the localization and biological behaviour of a tracer, a basic general scientific background is necessary. Several branches of knowledge, including chemistry, biochemistry, physiology, nuclear chemistry and physics, and pharmacokinetics, should be thoroughly mastered before the design of a new radiotracer is attempted. The effects of solute structures, binding proteins, and other components of the blood upon the radiotracer should be clearly understood. The appropriate approaches to the design of radiotracers must be based on informed comprehension of the influences of chemical structure, solubility, molecular or particle size, stability in vivo, as well as in vitro, and many other variables related to the application of radiotracers in humans. The problem, therefore, is the preparation of scientists having these combined skills. The lack of a program to prepare scientists to perform this task has stimulated us to create a course in radiopharmacology. The course is intended to cover, in a comparative, didactic, and practical fashion, those fundamental and clinical sciences that are the basis for the useful application of radiotracers in medicine. Lectures, laboratory exercises, and demonstrations form the core of the program, and opportunities to solve practical problems are provided to each individual. I believe this to be a pioneer program, unique in its conception and scope. This program provides graduate training in basic and applied radiopharmacology in order to render its participants capable of designing new radiotracers and understanding all the mechanisms involved in the use of radiotracers in biological research and in nuclear medicine. Candidates for this curriculum which leads to a Ph.D. degree in radiopharmacology, must have at least a master's degree in physics, biochemistry, or chemistry or a degree in medicine or pharmacology. The program is divided into two basic parts. The first part comprises a review of scientific knowledge applicable to the field of radiopharmacology. Depending on their previous training, the candidates take those courses necessary to complete the needed scientific background before entering the second part of the program. The basic courses that are mandatory in the first part are physics, chemistry (inorganic, organic, synthesis, etc.), anatomy and physiology, biochemistry, pharmacology, and toxicology. Mastery of these topics ensures that the basic knowledge and working tools are understood by everybody taking the second part of the program, in which the application of radiotracers in biology is taught. The length of the first part of the program will depend on the academic background of the candidate, and the courses can be taught as standard courses in different schools of the University. The second part of the program requires two semesters and is divided into five areas, each represented by several courses; 40% of the time is spent in lectures, conferences, and group demonstrations. The other 60% involves laboratory work in which mathematical problems have to be solved; different compounds have to be labeled with various radionuclides, including those of carbon, hydrogen, iodine, chromium, technetium, and other elements; animal experimentation must be mastered, including minor and major surgery in rodents, cats, and dogs; and proficiency must be acquired in tissue preparation for autoradiography and scintillation counting. The courses that make up the second part of the program are the following: 286
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A.
BASIC TECHNOLOGY
1. Mathematics algebra: exponentials; logarithms; proportions; analytic geometry; differential calculus; integral calculus; differential equations; dimensional analysis; probability and statistics. 2. Nuclear physics atomic and nuclear theory; contemporary atomic and nuclear physics; the radioactive nucleus: a, ~ and y decays; radioactive decay series; parent and daughter nuclides; production of radionuclides; acceleration of charged particles; detection of radioactivity; radionuclides for medical applications; dosimetry; radiation protection. 3. Nuclear chemistry chemical foundations of atomic theory; nuclear reactions: classification, production, and study; interaction of radiation with matter; biological effects of radiation; tracers in chemical applications; the radiotracer laboratory; radiochemical separations. B. RADIOTRACERS
1. Radiotracer design introduction; mathematical models; classical design concepts: pharmacological and physiological bases; design of binding sites. . Preparation concepts and criteria; tracer applications; radiolabeling, isotopic and non-isotopic; synthesis of 14Cradiotracers; biosynthetic procedures. . Chemical properties radionuclidic purity; radiochemical purity; carrier-free radiotracers; stability of radiotracers: chemical effects, radiolysis. 287
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4. Biological properties radiotracer receptors: molecular properties; radiotracer localization: ligand-receptor interactions; radiotracer interaction with hormone receptors, enzyme receptors, etc.; metabolism of radiotracers: stimulation and depression demonstration in vitro biological fate of radiotracers biological experimentation with radiotracers liquid scintillation and ~, counting, including tissue preparation autoradiography, including tissue preparation side effects of radiotracers data analysis in radiotracer studies: applications of computers absorbed radiation dose in radiotracer studies. 5. Biological transport general considerations; membranes: composition, structure, and function; transport of radiotracers: kinetics and thermodynamics effects of solute structure and binding proteins distribution of radioions and radiolabeled metabolites; techniques of studying the transport of radiotracers. 6. Mechanisms of localization a. Compartmental localization: theory and general considerations passive transfer: distribution of dilution localization by diffusion specialized transfer: active transport. b. Radiotracer study oJ cellular function individual organs or systems; brain: 14C-glucose; muscle: myocardium imaging; kidney: secretion and filtration; endocrine system: thyroid: trapping and metabolism of iodines other endocrine functions liver: hepatocyte function microsomal function: measurement in vivo skeletal system: radioion exchange in bone cells bone physiology demonstrated by radiotracer kinetics respiratory system: radioactive gases circulatory system: trapping and clearance of macroparticles clot formation and disposition of radiotracers.
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c. General processes o] radiotracer localization localization of free radioions in tumour cells; cell trapping of amino acids; phagocytosis and pinocytosis; other localizations observable in vivo; metabolism of radiotracers: demonstration in vitro; radiobioassays in demonstrating physiological changes: competitive protein-binding radioimmunoassay, etc.
THE FUTURE
OF RADIOPHARMACOLOGY
I believe that in establishing radiopharmacology as a new branch o f science, the first, most important steps have been taken. When the present efforts to organize scientists involved in this area are successful, I think that radiopharmacology will be able to stand by itself in the future. In order to spread the information gained by us, so others may take advantage of what has already been done and move toward the full integration of radiopharmacology into the biological sciences, very important steps have been taken. The first step has been the organization of the teaching program, that I described in the preceding pages; this program has proved invaluable in the preparation of scientists with the necessary skills to work in the development and applications of radiotracers. The second effort has been the organization of the First International Symposium on Radiopharmacology, which was held in Innsbruck, Austria, in May 1978. This symposium, sponsored by many important organizations, was extraordinarily successful. More than 380 scientists from 37 countries came to Innsbruck to participate in the symposium. The symposium concentrated mainly on an education program based on our teaching experience, and in which the fundamental aspects and biological applications of radiotracer technology were discussed. The symposium focused on the design and preparation of radiotracers, the interactions of radiotracers with binding sites, mechanisms of transport and localization of radiotracers, uptake and binding of radiotracers in the cells, fate of radiometabolites, and medical applications. Communications bearing on the transport and metabolism of radiotracers and the fate of radiometabolites were also presented. The interest shown by scientists in this symposium guarantees the success of the second international symposium, for which a site in the USA was chosen in a meeting of delegates from 34 countries during the Innsbruck symposium. The most recent step taken up to this time has been the organization of the International Association of Radiopharmacology, which has been functioning with temporary headquarters in Chicago. The association was founded after a worldwide poll was taken in which questionnaires were sent to more than 2,000 scientists from 48 countries. Results of the poll indicated almost 100% support for a second international symposium on radiopharmacology (only two responses were negative); 83.6% were in favour of creating the association. The main purpose of this association is to bring together all those interested in the uses of radiotracers for biological research and to organize the international and national meetings dedicated exclusively to this branch of science. The first central committee of the association includes the following members: Dr. Lelio G. Colombetti (Chicago), President; 289
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Dr. Kinichi Hisada (Kanazawa, Japan), Vice-President; Dr. Howard Glenn (Houston), Secretary; and Dr. W. Earl Barnes (Chicago), Treasurer. This committee is preparing a draft of the constitution and bylaws, which will be presented to all those who showed interest in the association. All scientists working with radiotracers for biological, including medical, applications are invited to become members of the association and join us in the development and uses of radiotracers for medical applications. W e believe that together we can make giant strides forward in developing all the tracers needed in medicine and in solving many biological problems that are today difficult to comprehend.
CONCLUSIONS "~Te attempted to present an objective view of the development of radiopharmacology and the importance of adapting an organized approach to the study of radiotracers in medical science. The use of radiotracers in diagnostic and therapeutics has been steadily increasing. Radiotracers have provided the medical science with a new methodology for studying the mechanisms of transport and action of drugs, have made possible the study of metabolism, and have helped tremendously in the prognosis of disease. ~Te foresee that teaching programs will be created in which scientists and physicians will be trained as researchers in this new area of science to fulfill the present and future needs.
SUMMARY The past, present and future development of radiopharmacology as a new branch of science is being presented. Radiopharmacological techniques have been applied by many scientists in the past, not knowing that they were developing a new scientific field. With the advancement of these techniques, the creation of a specialized teaching program and the organization of the first international symposium on radiopharmaeology, the first specialized meeting in this area, radiopharmacology became a discipline standing on its own feet. The main purpose of radiopharmacology is to study the chemical properties of radiotracers, and their interactions with living organisms. In order to promote and further expand this field, an international association of radiopharmacology was created. The main purpose of the association is to congregate all those interested in the uses of radiotracers in biological sciences, including medicine, by the organization of national and international meetings. REFERENCES 1. BRUCERM.A.: A tracer has no pharmacology - In: Vignettes in nuclear medicine. MaUinckrodt Nuclear Publ., St. Louis, Mo., 1966; chapt. 24. 2. CURm I., JOLIOXF.: Artificial production of a new kind of radioelement - Nature (Lond.) 153, 201, 1934. 3. GEXLXNGE. M. K., KELSEYF.E., MCINTOSHB.J., GANZA.: Biosynthesis of radioactive drugs using carbon-14 - Science I08, 588, 1948. 4. HEV~.SYVONG.: The absorption and translocafion of lead by plants - Biochem. J. I7, 439, 1923. 5. INTERNATIONALEncyclopedia of Pharmacology and Therapeutics (IUPHAR): COHENY. (Ed.): Pergamon Press, New York, 1971; 1st ed., forward section 78, vol. 1. 6. LEVmE R.R.: Pharmacology: drug actions and reactions - Little, Brown and Co., Boston, 1978; 2nd ed., p. 8. 7. McAFEE J.G.: Radioactive diagnostic agents: current problems and limitations - In: SUBRAMAmAN G., RHODES B.A.,. COOVER J.F., SODD V.J. (Eds): Radiopharmaceuticals. Society of Nuclear Medicine, New York, 1975; p. 3. 290
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8. NODINEJ.H., PLATT J.M., CARRANZAJ., DYKYJ R., MAPP Y.: Digital computer analysis of human isotopic drug kinetics - Int. J. appl. Radiat. 15, 263, 1964. 9. OKITA G.T., TALSO P.J., CURRY J.H., SMITH F.D., GEILING E.M.K.: Metabolic fate of radioactive digitoxin in human subjects - J. Pharmacol. exp. Ther. I15, 371, 1955. 10. ROTH L.J., LErER E., HOGNESS J.R., LANGHAMW. FI.: Studies of the metabolism of radioactive nicotinic acid and nicotinamide in mice - J. biol. Chem. t76, 249, 1948. 11. ULLBERG S.: Studies of the distribution of 35Sdabelled benzylpeniciUin in the body - Acta radiol. (Stockh.) 1IS(Suppl.), 1, 1954.
Requests/or reprints should be addressed to: LELIO G. COLOMBETTI Michael Reese Hospital and Medical Center Division o/ Nuclear Medicine 29th Street and Ellis Avenue Chicago, Ill. 60616 - USA
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