ABSTRACT

The isotopes of an element have the same number of protons in their atoms but different masses due to different numbers of neutrons. When a combination of neutrons and protons, which does not already exist in nature, is produced artificially, the atom will be unstable and is called a radioisotope. Radioisotopes can be manufactured in several ways. The most common is by neutron activation in a nuclear reactor. This involves the capture of a neutron by the nucleus of an atom resulting in an excess of neutrons. Some radioisotopes are manufactured in a cyclotron in which protons are introduced to the nucleus resulting in a deficiency of neutrons.

 The characteristics of an ideal radioisotope for therapy are quite different from those required for imaging.  For imaging, the radioisotope’s energy must be suitable for the camera crystal, without significant absorption by the tissue. Whereas, the energy of a therapeutic radioisotope must be deposited in the tissue to damage the DNA chains to prevent the diseased cells from replicating.

 Iodine is a nonmetallic solid element. There are thirty three known isotope of iodine from ¹¹⁰I to ¹⁴²I all of them are radioactive except the stable iodoine-127. Iodine is among the most widely used radionuclides, mostly in the medical field, because of its rather short half-life and useful beta emission from some of its isotopes. Among the thirty-two radioactive iodine isotopes, four of them are of special interest, namely,  ¹²³I, ¹²⁴I, ¹²⁵I and ¹³¹I.

 Radioisotopic labeling is a technique for tracking the passage of a chemical substance through a system. The chemical substance is "labeled" by including radionuclides in its chemical composition. When these decay, their presence can be determined by detecting the radiation they emit.

 A radiopharmaceutical may be defined as a chemical or pharmaceutical preparation labeled with a radionuclide in tracer or therapeutic concentration, used as a diagnostic or therapeutic agent. A radiopharmaceutical agent is usually administrated into a vein. Depending on which type of scan is being performed, the imaging will be done either immediately, a few hours later, or even several days after the injection. Imaging time varies, generally ranging from 20 to 45 minutes.

 The active ingredient, that is the actual radiolabeled chemical, is usually present in minute amount, typically less than a nanogram and far below the amount necessary to initiate any physiological response in an individual. The radiopharmaceutical that is used is determined by what part of the body is under study, since some compounds collect in specific organs better than others.

In this thesis, we are more interested in the central nervous system (CNS) which is responsible for the rapid regulation of the functions of the various systems of the body according to the changes in the external and internal environments of the body.

 Among the CNS receptors, 5-Hydroxytryptamine is reported to be very important with respect to the study of brain disorders. The distribution and the density of these receptors have direct links with some CNS disorders such as anxiety, depression and Alzheimer’s disease.

 Another type of CNS receptors is dopamine receptors. There is now considerable evidence for the existence of at least five subtypes of dopamine receptors. The human dopamine system is involved in the regulation of brain functions as controlling motor. Disruptions of its functioning have been implicated in neurological and psychiatric illnesses.

 In-vivo imaging of serotonin and dopamine receptors is therefore expected to contribute greatly to both the understanding of the human brain mechanisms and the treatment of psychiatric disorders.

 In general, the work in this thesis is related to the labeling of two biologically active organic compounds with radioactive ¹²⁵I aiming to image serotonin and dopamine receptors located in the brain.

Chapter I: Radiosynthesis and Biological Evaluation of ¹²⁵I-Metergoline as Serotonin (5-HT) Receptor Radioligand

In this chapter we studied the labeling of metergoline, (MG) [([[(8β)-1,6-Dimethylergolin-8-yl]methyl] carbamic acid phenylmethyl ester), an ergot derivative with high affinity for 5-HT receptors and as antagonist for 5-HT₁, 5HT₂ and 5HT₇ receptor] with radioactive iodine which may be useful as a serotonin receptor prop.

This chapter is divided into three parts:

1. The first part is an introduction concerning the metergoline , its importance and a study review on the metergoline.

2. The second part contains detailed information concerning the chemicals, reagents, the radionuclide, the equipment and the counting systems used in the study. It describes the electrophilic radioiodination using oxidizing agents.

3. The third part deals with the labeling of metergoline  by electrophilic substitution which was prepared by using a simple procedure, and the biodistribution of the labeled product in mice.

 The obtained results showed that:

1. The labeled Metergoline was obtained by using a simple ethanolic solution of metergoline which was mixed with chloramine-T and the pH was adjusted to 4, then the radioactive iodine was added. The reaction mixture was incubated at room temperature (25 ºC) for 15 minutes. The radiochemical yield reached 97 %.

2. The effect of the hydrogen ion concentration on the radiochemical yield of ¹²⁵I-MG revealed the flexibility of the labeling reaction as the radiochemical yield was high along the pH range.

3. The amount of metergoline, in the range of 100-400 μg, had almost no effect on the radiochemical yield of ¹²⁵I-MG. 

4. The effect of the amount of chloramine-T on the labeled metergoline revealed that by increasing the amount of chloramine-T from 50μg to 200μg, the radiochemical yield of the labeled metergoline was always high.

5. High radiochemical yield of ¹²⁵I-MG was obtained by incubating the reaction mixture at room temperature. Increasing the reaction temperature beyond the room temperature had no effect the radiochemical yield.

6. The reaction time had a slight effect on the radiochemical yield of ¹²⁵I-MG; the optimum time was 15 minutes.

7. The in-vitro stability test of the labeled metergoline shows that the ¹²⁵I-MG was stable for 24 hours which is an enough time for the imaging of the brain.

8. The initial biodistribution studies revealed the accumulation of ¹²⁵I-MG tracer in the brain tissues. It was retained for long time with a percentage sufficient to image serotonin receptors in the brain (1 %/g at 60 minutes post injection). 

9. The in-vivo binding affinity of the ¹²⁵I-MG tracer was measured by blocking the receptors with clozapine that reduced the tracer uptake by the brain markedly.

Conclusion: These findings showed that ¹²⁵I-MG tracer displays the desirable properties as an agent for imaging the serotonin receptors located in the brain.

Chapter II: Radiosynthesis and Biological Evaluation of [¹²⁵I]-Spiperone as D2-Dopamine Receptor Radioligand.

In this chapter we studied the labeling of the spiperone (SP) which is a selective D2-dopamine receptor antagonist. This chapter includes:

1. The first part is an introduction concerning the spiperone, its importance and a study review on the spiperone. 

2. The second part contains detailed information concerning the chemicals, reagents, the radionuclide, the equipment, and the counting systems used in the study. It describes the electrophilic radioiodination using oxidizing agents.

3. The third part deals with the labeling of spiperone by electrophillic substitution which was prepared by using a simple procedure and the biodistribution of the labeled product in mice.

The results showed that:

1. The spiperone was labeled with iodine-125 in a neutral pH 7,  using chloramine-T as an oxidizing agent, via heating the reaction mixture at 70 ºC for 15 minutes. The obtained radiochemical yield was 96%.

2. The effect of the pH of the reaction mixture on the radiochemical yield of ¹²⁵I-SP revealed that high yield of 97% was obtained at neutral pH 7, and that the radiochemical yield decreased in acidic or alkaline media.

3. The effect of the amount of spiperone on the radiochemical yield proved that a small amount of spiperone yielded low radiochemical yield of ¹²⁵I-SP. Increasing the amount of spiperone above 50μg produced a high radiochemical yield which exceeded 95 %.

4. The effect of the amount of chloramine-T on the radiochemical yield of ¹²⁵I-SP revealed that the yield increased by increasing the chloramine-T concentration up to 150μg. Increasing the amount of chloramine-T to 350 μg caused a decrease of the radiochemical yield of ¹²⁵I-SP.

5. The effect of the reaction temperature on the yield of ¹²⁵I-SP revealed that increasing the reaction temperature to 70°C, resulted in an increase in the radiochemical yield of ¹²⁵I-SP to 96.5%. Rising the temperature above this value caused a fast decrease in the radiochemical yield.

6. The effect of the reaction time on the radiochemical yield of ¹²⁵I-SP revealed that increasing the reaction time to 10 minutes caused an increase in the radiochemical yield to 96%.

7. The effect of the solvent type on the radiochemical yield did not show any significant variation in the yield of ¹²⁵I-SP as all the solvents used were able to dissolve spiperone.

8. The in-vitro stability test of the labeled spiperone proved that ¹²⁵I-SP was stable for approximately 8 hours which is an enough time for the imaging of the brain.

9. The in-vivo biodistribution studies showed that the initial brain uptake correlated fairly well with the brain uptake index and that the kinetics of the radioactivity specifically bound to the striatum were strongly influenced by the dopamine receptor binding affinity of the compound.

10. ¹²⁵I-Spiperone binded with high affinity to dopamine receptors in-vivo. Specific binding is about 65 percent of total binding as is displaced stereo-specifically by clozapine.

Conclusion: ¹²⁵I-Spiperone may prove to be a useful ligand in the studies of examining D2-dopamine