Benha University Faculty of Science Chemistry Department

RADIOCHEMICAL STUDIES ON THE SEPARATION OF 131 AND RADIOIODINATION OF SOME ORGANIC COMPOUNDS

A Thesis Submitted by MAHMOUD ABBAS ISMAIL MOHAMED and Radioactive Generators Dep., Hot Labs. Center Atomic Energy Authority

To Faculty of Science – Benha University

Presented as partial fulfillment of The Degree of M.Sc In chemistry

Supervised by

Prof .Dr .H .A. Dessouki Prof . Dr .S .A .El Bayoumy Prof. of Inorganic and Prof. of Radiochemistry Analytical Chemistry Isot. and Radio Generators.Dept, Faculty of Science Benha Univ. Atomic Energy Authority

Dr. Shoukar T.M. Atwa Dr. A.A. ElMohty Lect. of Physical Chemistry Pro Ass it. Prof.of Radiochemistry Faculty of Science Benha Univ. Isot. and Radio Generators.Dept,

Atomic Energy Authority

2010 آ ام اء

درات آ إ اد ١٣١ و اآت ا د ا

ر

ــــد س ا ا واات ا – آ ا ارة ه ا ار

ا آــ اــــ ــم – ــ

ل در ا اء

اـــــــــاف

د.أ / ا ا د.أ / د اذ اء ا و ا أذ اــء اـــ آ ام ـــ ا واات ا هــــ اـــ ارــــــــ

د / ر .م.أ د / أ ا رس اء ا أذ اــء اـــ ا واات ا آ ام ـــ هــــ اـــ ارــــــــ

٢٠١٠ List of abbreviations

Abbr. Referent CAT ChloramineT H2O2 peroxide HPLC High performance liquid chromatography TLC Thin layer chromatography Temp. Temperature Conc. Concentration min Minute NCA No Carrier Added Rf Relative front Rt Retention time CNS Central nervous system Yindole 4[2hydroxy3 (isopentylamino)propoxy] indole

Epidepride N[(1ethyl2 pyrrolidyl)methyl]2,3 dimethoxy5(tributylstannyl) benzamide

SPECT Single Photon emission computed tomography

PET Positron emission tomography HPGe High purity cpm count per minute ETRR2 The second Egyptain Research Reactor h Hour Micro β Beta particles α Alpha particles γ Gamma emission d Day g gram Ci Curie (radioactivity unit) 131mTe in another energy state KeV kilo electron volt Av average a.m.u unit EC (n,f) neutron fission reaction 123 IBZM [123 I]Iodine substituted benzamide At ICl Iodine monochloride MW Mega watt

ACKNOWLEDGMENT

This work was carried out in the laboratories of the Isotopes and Radioactive Generators Department, Isotope Production Division, Hot Labs. Center, Atomic Energy Authority, Inshas, Egypt.

The author wishes to thank deeply Prof. Dr. Hassan Ali Dessouki, Professor of Inorganic and Analytical Chemistry, Faculty of Science, Benha University, for sponsoring this work and for his valuable and constant interest throughout these studies.

I would like to express my deep gratitude and sincere appreciation to Prof. Dr. Sami AbuBakr El Bayoumy Isotopes and Radioactive Generators Department, Atomic Energy Authority, to whom I am greatly indebted for his great help, encouragement, valuable and sincere efforts in reviewing this thesis.

The author would like to thank Prof. Dr. Ahmed AbdElMohty Bayoumy, Head of Isotopes and Radioactive Generators Department, Atomic Energy Authority, for effective supervision advice, continuous encouragement through the whole work.

My deep and profound gratitude to Dr. Shoukar Tawfik Faculty of Science, Benha University, for her sincere efforts during the experimental work and revision of this thesis.

The author expresses his gratitude and sincere appreciation to Dr. Ahmed Ali AbdElSadek, Isotopes and Radioactive Generators Department, Atomic Energy Authority, for his helpful and valuable guidance during the experimental work.

The author would like to thank Dr. Mahmoud Hamdi Mahmoud Sanad, Labeled Compounds Department, Atomic Energy Authority, for continuous help.

To

My parents

My wife

My sons ( Mennh, Tasneem)

My best friends

Approved Sheet

Title: RADIOCHEMICAL STUDIES ON THE SEPARATION OF IODINE131 AND RADIOIODINATION OF SOME ORGANIC COMPOUNDS

Name : MAHMOUD ABBAS ISMAIL MOHAMED Isotopes and Radioactive Generators Dep., Hot Labs. Center Atomic Energy Authority

Supervisors :

Name Position Signature Prof. Dr .H .A. Dessouki Prof. of Inorganic and Analytical Chemistry Faculty of Science Benha Univ.

Prof . Dr .S.A. ElBayoumy Prof. Prof. of Radiochemistry Isot. and Radio Generators.Dept, Atomic Energy Authority

Dr. A.A. ElMohty Assis. Prof.of Radiochemistry Isot. and Radio Generators.Dept, Atomic Energy Authority

Dr. Shoukar T.M. Atwa Lect. of Physical Chemistry Faculty of Science Benha Univ.

Head of Chemistry Department

Prof. Dr. S.G. Donia

2010

ا ا د س ا ان ا درات آ ا اد ١٣١ و اآت ا د ا ار ا اء ا

« ﻟﺠـﻟﺠﻟﺠﻟﺠ ﻨـــﻨﻨﻨ ــــــ ﺔ ﺍﻹﺔ ﺍﻹ ﺷ ــــــ ﺮﺍﻑ »

ا ا ا د.ا / د اذ اء ا آام .ا د / أ ا اذ اء ا ه ا ار .ا د / أ ا اذ اء ا ه ا ار د / آر رس اء ا آ ام

ر اء

د.ا( / ل د)

٢٠١٠

ا ا د س ا ان ا درات آ ا اد ١٣١ و اآت ا د ا ار ا اء ا

« ﻟﺠـﻟﺠﻟﺠﻟﺠ ﻨـــﻨﻨﻨ ــــــ ﺔ ﺍﻟﻤﻨ ـــ ﺎﻗﺸﺔ »

ا ا ا د.ا / رأ د اذ اء ا ر او م ا د.ا / اذ اء ا ه ا ار د.ا / د اذ اء ا آام .ا د / أ ا اذ اء ا ه ا ار

ر اء

د.ا( / ل د)

٢٠١٠

Contents

Page Chapter I I. Introduction.………………..………………………….………..…...…...1 I.1. General Considerations ……………….……………………...... …...1 I.2 Physical and Chemical Properties of ……...... 3 I.3 Physical and Chemical Properties of Iodine...... …...... 3 I.4. Radiochemistry of Iodine……………….…………………..….……...6 I.5 Production of 131 I………………………………………………..……..10

I.6 Types of nuclear reactions……………………………………...... ….11 I.6.1 (n,γγγ) reactions.…………………………………………………..…...11 I.6.2 Multistage "double neutron" (2n,γγγ) reactions…...……………..….12

I.6.3 (n,p) reactions………………………………………………………..13 I.6.4 (n,ααα) Reactions..…………………………………………………..…13 I.6.5 Fission reactions………………………………...…………………...13

I.7 Definition of the labeled compound .…………………..…………...…14

I.8 Types of labeling .…………………………………………………..…..14

I.9 Methods of labeling Pharmaceutical compounds………………...…14 I.9.1 Chemical synthesis…..…….………………………………………...14 I.9.2 Biosynthesis…..…...…………………………………………………15 I.9.3 Recoil labeling……….………………………………….……...... ….15 I.9.4 Excitation labeling….………………………….…………………....16 I.9.5 Bifunctional chelates labeling.…….………………..…………...... 16 I.9.6 Isotopic exchange reactions…….……………….……………...... 16 I.10 Radiopharmaceuticals.………………………..…….….……....…....17 I.11 used for diagnostic nuclear medicine….………...…18 i

I.11.1 Single photon emission computed tomography (SPECT)……….18 I.11.2 Dual and triple head camera…………………………...…………19 I.11.3 Positron emission tomography (PET)……………………….……19 I.12 Preparation of radioiodinated compounds…………….………..….21 I.12.1 Electrophilic Substitution………….………...……….…………...22

I.12.1.1 ChloramineT (CAT) method………….…..………..…………..22 I.12.1.2 Iodogen method…………………………..………………….…...23 I.12.1.3 Enzymatic method…………………………..…………….……..24 I.12.1.4 Electrolytic method………………………………………………25 I.12.2 Nucleophilic substitution…………………………………….….…25 I.12.2.1 Exchange in solvent………………...…………………………....26

I.12.2.2 Exchange in melt…………………………………………………26 I.12.2.3 Exchange for ………………….………………………..27 I.12.2.4 Catalyzed exchange …………...………………………………...28

I.13 Purification of radioiodinated compounds...... 28 I.14 Quality control ...... 29 I.14.1 Chemical purity ………………………………..……………….…30 I.14.2 Radionuclidic purity ………….…………………………………...30

I.14.3 Radiochemical purity………………………………….…….…….31 I.15 Apyrogenicity, Toxicity and Biological distribution…….….…….. 32

Chapter II II.Experimental…...…………………………………….…………..…….33

II.1 Materials……….…..…………………………………………………33 II.2 Equipment…..…..…..……………………………….………………..33 II.3 Preparation and irradiation of target………………………………36

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II.4. Factor affecting the production of 131 I……………………..……….36 II.4.1 Effect of oven temperature on the releasing 131 I activity.………..36 II.4.2 Effect of distillation time on the releasing 131 I activity and the concentration of tellurium in the separated radioiodine….…37 II.4.3 hydroxide concentration……..…..……………………….37 II.5 Quality control on the released 131 I.……………………………...….37 II.5.1 Chemical Purity…………………………………...………………..37 II.5.2 Radionuclidic Purity………….……...……………...………..……39

II.5.3 Radiochemical purity….………………...…………………………39 II.6 Iodination of 4[2hydroxy3(isopentylamino) propoxy] indole

(Yindole)...... 40 II.6.1 Cold iodination of Yindole………………………………………40 II.6.1.1 ChloramineT method………………………………………..….40 II.6.1.2 Iodogen method…….……...……………………………….….....41 II.6.2 Radioiodination of Yindole…………………………………………..41 II.6.2.1 ChloramineT method………………………………………………41 II.6.2.2 Iodogen method……………………………………………………...42 II.7 Iodination of [(1ethyl2pyrrolidyl) methyl] 2,3dimethoxy5(tributylstannyl) benzamide (Epidepride)….….42 II.7.1 Cold iodination of (Epidepride)………………………………….43 II.7.1.1 ChloramineT method……...………………………...... 43 II.7.1.2 Hydrogen peroxide method...…………….……………...... …..44 II.7.2 Radioiodination of epidepride……………..………….…………...44

II.7.2.1 ChloramineT method…………………………..……………….44 II.7.2.2 Hydrogen peroxide method………………………….………….44 II.8 Biological distribution of the labeled compound in mice...... 45

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Chapter III III.Results and Discussion………………………...….…………………..46

III.1 Irradiation of target material………………………………………46 III.2 Dry distillation method………………….………………………….49 III.2.1 Effect of oven temperature on the releasing 131 I activity……….49 III.2.2 Effect of distillation time on the releasing 131 I activity and on the tellurium concentration in the separated radioiodine .…….49 III.2.3 Effect of sodium hydroxide concentration….…….………..……52 III.3 Quality control of the produced 131 I………………………………..52 III.3.1Chemical purity……………………………………………………52 III.3.2 Radionuclidic purity………………………………...…...………..53 III.3.3 Radiochemical purity……………………………………….…….56 III.4 Optimization of production of 131 I ………………………...……….56

III.5 Radioiodination of Yindole...……………………………....………59 III.5.1 introduction .………………..……………………………..….…...59 III.5.2 Chromatographic identification of 131 IYindole……………....60 III.5.3 Factors affecting the radiochemical yield………………………...64 III.5.3.1 Effect of Yindole concentration………………….…………….64 III.5.3.2 Effect of pH of the medium…………..……………...…………..64 III.5.3.3 Effect of oxidizing agent concentration ...………………………66 III.5.3.4 Effect of reaction time…….……………………………………..66 III.5.4 Optimum conditions suggested for the preparation of pure 131 IY indole...... 67 131 Invitrostability of the labeled IYindole…………………………….69 131 III.5.5 Biodistribution of IYindole in mice.……………...……....….69 III.6 Radioiodination of Epidepride ………………………………….…72

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III.6.1 introduction………………………………………………………..72 131 III.6.2 Chromatographic identification of Iepidepride………..…….74 III.6.3 Factors affecting the radiochemical yield…………………….…..77 III.6.3.1 Effect of epidepride concentration……………………………...77 III.6.3.2 Effect of pH……………………………...…………………….....79 III.6.3.3 Effect of reaction time………………...…………………………79 III.6.3.4 Effect of oxidizing agent concentration………………...………81 III.6.4 Optimum conditions suggested for the preparation of pure 131 I epidepride……………………………………………………….….....82 III.6.5 Invitrostability of the labeled 131 Iepidepride……...... 82 References…………………………………………………………………85 English summary…………………………………………………….xxiii

١-٤ ...………………………………………………………Arabic summary

v

List of Figures

131 Fig. (1): Production of I………...... 10 Fig. (2): Schematic diagram of the distillation apparatus used for 131 production of I from irradiated TeO 2 target…………………...……35

Fig.(3): Standard absorption curve of tellurium……………………….39

Fig. (4) γ ray spectrum of thermal neutrons irradiated TeO 2 target material measured after 48 h from the end

of irradiation………………………………………………....…48 Fig. (5): The effect of oven temperature on the releasing 131 I activity……………………………………………………..51 Fig. (6): The effect of time and oven temperature on the Released 131 I activity………………………...…………………51 Fig.(7): Effect of NaOH concentration on sorption of the produced 131 I…………………………………………….54 131 Fig.(8): γ ray spectrum of the produced I ………………...…...…...55 131 Fig.(9): Decay curve of the produced I ………...…….……...………55 131 Fig.(10): Radiochromatogram of the produced I……………..……...57 Fig.(11):Schematic flow sheet of the 131 I production Process via dry distillation technique………………………………………..58 Fig. (12):UV Absorbance of KI after separation on RP18 HPLC column using 0.1M sod. bicarbonate: acetonitrile (1:1) at a flow rate 1 ml/min……………..………………………….…..62 Fig. (13): UV Absorbance of Yindole after separation on RP18 HPLC column using 0.1 M sod. bicarbonate: acetonitrile

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(1:1) at a flow rate 1ml/min………………………….…………62

Fig. (14): UV Absorbance of mixture of KI and Yindole after separation on RP18 HPLC column 0.1M using sod. bicarbonate: acetonitrile (1:1) at a flow rate 1 ml/min...... 63 Fig.(15): Variation of the radiochemical yield of [131 I] iodoYindole with the concentration of Yindole………………………..…...…..65 Fig.(16): Effect of pH on the radiochemical yield of [131 I] iodoY indole…………………………………………………………..65 Fig.(17): Variation of the radiochemical yield of [131 I] iodoYindole with the concentration of oxidizing agents…………………….…….68 Fig.(18): Effect of reaction time on the radiochemical yield 131 of [ I] iodoYindole ….……………………………………….68 Fig.(19): HPLC analysis of the reaction mixture of [131 I]iodoY indole………………………………………….………...………70 Fig. (20): UV Absorbance of KI after separation on RP18 HPLC column using 0.1 M phosphate buffer pH 7.5: ethanol (30:70) at a flow rate 1 ml/min……………………………...... 75 Fig. (21): UV Absorbance of epidepride after separation on RP18 HPLC Column using 0.1 M phosphate buffer pH 7.5 : ethanol (30:70 at a flow) rate 1 ml/min……………………....75 Fig. (22): UV Absorbance of epidepride after separation on RP18 HPLC Column using 0.1 M phosphate buffer pH 7.5:

ethanol (30:70) at a flow rate 1 ml/min...... 76

Fig.(23): Effect of tributyltin epidepride concentration on the radiochemical yield of [ 131 I] iodoepidepride using

H2O as oxidizing agent……………...... ……….. 78 vii

Fig.(24): Effect of pH on the radiochemical yield of 131 [ I] iodoepidepride using H2O2 method……………………...80 Fig.(25): Effect of the reaction time on the radiochemical yield of [131 I] iodoepidepride ..……………………………………….…80 Fig.(26): UV absorbance of the cold reaction of KI, tributyltin epidepride and CAT after separation on RP18 HPLC column using 0.1 M phosphate buffer pH 7.4: ethanol (30:70) as eluent at a flow rate 0.5 ml/min………………..……………....84 Fig. (27): HPLC analysis of the reaction mixture of [131 I] iodoepidepride …………………………...………..….84

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List of Tables

Table (1): The physical properties of tellurium...... 3

Table (2): The physical properties of iodine...... 4

Table (3): Radioisotopes of iodine and their nuclear characteristics...... 7

Table (4): Isotopes of natural tellurium and their abundance…………..9

Table (5): Routine methods of production of some commonly

used γray emitters...... 20

Table (6): chromatographic separation of radioiodine species...... 32

Table (7): The concentration of tellurium in the separated

radioiodine at optimum temperature 700 ◦C…………..……52

Table (8): Summary of 131 I production via dry distillation technique…56

Table (9): Invitrostability of [ 131 I] Yindole...... 71

Table (10): Biodistribution of [ 131 I] iodo –Y indole...... 71

Table (11): Table (10): Variation of the radiochemical yield of [131 I]

iodoepidepride with the oxidizing agents concentration….82

131 Table (12): Stability of [ I] iodoepidepride………………….…………83

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SUMMARY

This thesis is constituted of three chapters :

Chapter I

It deals with the theoretical consideration of the subject. The chapter deals with the importance of radioisotopes in medical applications, and the physical and biological properties of these isotopes. Also, this part deals with the chemical and physical properties of both tellurium and iodine and the methods of the production of radioiodine from tellurium targets especially dry distillation method and ion exchange method. It deals with general methods of labeling, chemistry of iodine especially the most frequently used in nuclear medicine, their methods of production and applications. It includes also the techniques used for the preparation of the radioiodinated compounds, especially the electrophilic technique or the oxidative radioiodination technique. In this technique, oxidizing agents are used to oxidize iodide ions to iodonium ions capable of electrophilic attack on the aryl of the organic compound. This chapter deals also with the receptor tracers, their types and the effects that can occur due to the binding of these receptors to the cell membrane. Since these radiopharmaceuticals are used for diagnosis and therapeutic treatment of human diseases, quality control tests such as chemical purity, radionuclidic purity, radiochemical purity, sterility, apyrogenicity and biodistribution are performed to ensure the purity, the safety and efficiency of these products for the intended nuclear medicine application.

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Chapter II

It contains detailed information concerning the chemicals, reagents, the radionuclides, the equipment and the counting systems used in the study. It describes production technique of iodine131 using dry distillation method. It describes also the electrophilic radioiodination for each of Yindole and epidepride. Analysis of the labeled products was performed using two chromatographic techniques. The first technique is thin layer chromatography in which the compound was identified by its retardation factor (R f). The second technique is the purification of the labeled compounds by means of high pressure liquid chromatography (HPLC). On the reverse phase RP18, column eluted with an appropriate solvent where each compound is identified by its retention time (R t).

Chapter III

It deals with the idea of separation of iodine131 from tellurium targets using the dry distillation method. The factors affecting the production and separation of 131 I were studied such as the oven temperature; the distillation time and the sodium hydroxide concentration, the following flow sheet summarizes the production process. The quality control tests were carried out on the final product (Na 131 I). They were 99.9%, 98.7% for radionuclidic purity and radiochemical purity, respectively and the chemical purity of Te in the final radioiodine was 0.8g/ml.

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5 g TeO 2

Irradiated at thermal neutron flux of 14 2 10 n/cm .s for 12 h

131 mTe, 131 Te, and 131 I

(Irradiated TeO target) 2

Dry distillation at 700 ◦ C Separated 131 I 5M H 2SO 4 Condenser for 30 min vapors

5 ml 0.1M NaOH

131 Quality control Produced I

Radionuclidic Radiochemical Chemical

Schematic flow sheet of the 131 I production process via dry distillation technique. 131 The produced I was used for radioiodination of two medically important compounds namely, 4[2hydroxy3(isopentylamino) propoxy] indole (Yindole) and N[(1ethyl2pyrrolidyl) methyl]2, 3dimethoxy (tributylstannyl) benzamide (Epidepride) . Radioiodination was carried out by using several oxidizing agents such as chloramineT; iodogen and

xii

hydrogen peroxide. They are used to oxidize iodide to the iodonium which attacks the aryl group of the compound to be labeled. The factors affecting the radiochemical yield such as substrate concentration, pH, oxidizing agent concentration and reaction time were extensively studied. The conditions, which gave high radiochemical yield, were summarized in one reaction to give the optimum radiochemical yield. Optimization of the radiochemical yield resulted in 50% for 131 IYindole was obtained when the reaction was carried out in 0.1 M phosphate buffer (pH 7) for 30 min. A radiochemical yield of 95% for 131 Iepidepride when the reaction was carried out at pH 3.4 for 20 min. Purification of the labeled compounds was performed resulting in 99% radiochemically pure products. The stability of the purified labeled compounds was carried out to determine the suitable time for injection at which no side products due to radiolysis can occur. The biodistribution of [131 I] iodoYindole was performed in mice. The data shows that the radioactivity localized in brain, heart and lung were 1.28%, 4.6% and 2.3%, respectively at 45 min post injection.

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Introduction

I.1 General Considerations Nuclear reactors, cyclotrons and accelerators are important sources of artificial radioactive isotopes which opened a new field of tremendous importance (Wiza and Szymilewiez, 1985). Radioactive isotopes have been found valuable applications in the field of chemistry, medicine, industry , agriculture and biology (Sachdev et al., 1999). Production of high specific activity radioisotopes especially carrierfree ones from materials irradiated in nuclear reactors represents an important field of nuclear and radiochemistry (Wei et al., 2000). The choice of radioisotopes for medical applications require an accurate knowledge of the nuclear structure and decay data which are needed for selecting a particular radioisotope for invivo medical applications (Qaim, 1982). The selection is determined by the following two factors; 1 Resolution and efficiency of the radiation detecting system used. 2 Radiation dose caused to the patient. The following points should be kept in mind while selecting a target material for irradiation in a reactor (IAEA, 2003). • Substances which are explosive, pyrophoric, or volatile, are not to be irradiated in the reactor. • Target should be stable under irradiation conditions. • High purity targets should be irradiated. • Enriched targets should be materials which will enable to produce of radioisotope with high specific activity, physical form of target and/or target geometry should be minimum. • Target should be in a suitable chemical form to enable post irradiation

1

processing. Usually target in metallic form or oxides are preferred. The shortlived radioisotopes with halflives of no more than few hours, especially γemitting ones as, 137mBa and 113mIn have attracted considerable interest in diagnostic nuclear medicine applications (Lambrecht, 1983). The βemitters isotopes such as 153 Sm, 186 Re, 188 Re, 166 Ho and 131 I have a significant application in radiotherapy methods as; radioactive intraarterial microspheres, chemicallyguided bone agents, labeled monoclonal antibodies and isotopicallytagged polypeptide receptor binding agents (Kerting et al., 1997). The most promising among these radionuclides is 131 I. This has favorable nuclear and physical characteristics where, its γray energy of 364 KeV with halflife of 8.02 days is suitable for scanning and detection outside the body, but it is much more penetrating and requires increased protection for the people involved in the synthesis and the use of radioiodinated compounds. The βradiation

131 dose [ β max = 606.3 KeV (89.4%)] of I given to the patients must be minimized but this medium energy can kill cells and it has proven to be useful in the radiotherapy of thyroid cancer (Humm, 1986). Its radiotherapeutic potential and its very good commercially availability made it an attractive radionuclide for radiotherapy. In particular it allows studying some slow biochemical kinetics of iodinated compounds via Positron Emission Tomography (PET). (Herzog et al., 2002). 131 I and 123 I radioisotopes have been widely used in the labeling of monoclonal antibodies (Bourdoiseau, 1986).

2

I.2 Physical and chemical properties of tellurium Tellurium is silverywhite semi metallic. It is located in group (VI) of the periodic table that contains S, Se, Te and Po. The four elements are nonmetals. They are called or oreforming elements, because a large number of metal ores are oxides or sulphide. The solar system content of tellurium is ∼ 1.57 X 10 8 % where, earth crust content is ∼ 0.001 mg/Kg (Lide, 1997). The physical properties of tellurium is summarized in Table (1)

Table (1) The physical properties of tellurium. Average Atomic Ionization Density Melting point Boiling point mass energy (eV) (g/cm 3) (◦C) (◦C) (a.m.u) 127.60 9.01 6.24 733 988

Tellurium has the electronic structure [Kr] 4d10 , 5s2, 5p4. Tellurium has the following oxidation numbers of (II), (+II), (+IV) and (+VI) (Haaland, 1995).

Tellurium dioxide (TeO 2) is a non volatile white salt; its melting point o is 733 C. It is made by burning tellurium in air. TeO 2 is almost insoluble in water but dissolves in strong bases to form tellurites, acid tellurites and various poly tellurites. It also dissolves in acids to form basic salts: this illustrates the amphoteric character of TeO 2. It is advisable to take care that 131 the target material is TeO 2 for production of I (Drake, 1994).

I.3 Physical and chemical properties of iodine. Iodine occurs as iodide in the form of sodium and iodate. Also, iodine concentrates in various forms of marine life. Iodine is a black

3 solid with a slight metallic luster. At atmospheric pressure it sublimes giving a violet vapor. Its solubility in water is slight (0.33g/L at 25 ◦C). It is readily soluble in nonpolar solvents such as CS 2 and CCl 4 to give violet solutions. (Bellucci, 1995). The solar system content of iodine is 2.9 X 10 9 % where, earth crust content is ∼ 0.45 mg/Kg and sea water content is ∼ 0.06 mg/L ((Lide, 1997). The physical properties of iodine are shown in table (2).

Table (2) The physical properties of iodine. Av. Atomic mass Ionization Density Melting Boiling Specific (a.m.u) energy (eV) (g/Cm 3) point ( ◦C) point ( ◦C) heat (J/g) 126.90 10.45 4.93 113.7 185.2 0.145

Iodine is located in group (VII) of the periodic table that contains F, Cl, Br, I and At. Iodine has the electronic structure [Kr] 4d10 , 5s2, 5p5. It gains an electron by forming an ionic bond (X ) or a covalent bond to complete its octet. The properties of and bromine are closer than those between the other pairs of elements because their sizes are closer. The oxidation states (+I) and (I) are the most common. Higher oxidation states (+III), (+V) and (+VII) exist for all of the elements except F. The higher valence states arise quite by promoting electrons from filled P and S levels to empty d levels which are found in oxides. Iodine atom is the largest in the VII group. The increased size results in less effective overlap of orbital, this trend is observed with Cl 2, Br 2 and also with and I 2. All the elements exist as diatomic molecules, and they are all colored .Gaseous F 2 is light yellow, Cl 2 gas is yellowgreen, Br 2 gas and liquid are dark redbrown, and I 2 gas is violet. The colors arise from the

4 absorption of light on promoting an electron from the ground state to higher state. Solid I 2 crystallizes as black flakes. Liquid I 2 conducts very slightly because of its selfionization.

+ 3I2 I 3 + I 3

There are two different commercial methods for obtaining iodine. The mainly method depends upon the use of chill saltpeter source which mainly consists of sodium nitrate NaNO 3 with trace amounts of sodium iodate

NaIO 3 and sodium periodate NaIO 4.

2 + 2IO 3 + 6HSO 3 2I + 6SO 4 +6H + 5I + IO 3 + 6H 3I2 + 3H2O

Small amounts of iodine are required in the human diet, so traces (10ppm) of NaI are added to table salt. KI is added to animal poultry feeds. The thyroid gland produces a growth regulating hormones called thyroxine and triiodothyronine which contains iodine. Deficiency of iodine causes the goiter disease. In the laboratory iodides and iodate are used in volumetric , analysis, and Nessler s reagent K2[HgI 4] is used to detect ammonia. Iodine is a weaker oxidizing halide, because it does not easily react with water and the reaction is endothermic. The free energy change is positive, which shows that the energy must be supplied (105 KJ/mol) to make it oxidize water.

+ I 2 + H 2O 2H + 2I + 1/2 O 2

5

The reverse reaction would be 105 KJ/mol, so the reverse reaction should occur spontaneously (Cotton, 1999). Iodine oxides are the most stable of the halogen oxides. The higher oxidation states are the more stable than the lower states. The bonds are largely covalent because of the small difference in the electro negativity between the halogen atom and atom, but I 2O4 and I 4O9 are ionic (Kraft, 1995). Iodine forms a number of species in aqueous solutions, ranging from the (–1) oxidation state (iodide) to +7 (periodate). Microscopic studies indicate that dimerization occurs in solutions to some extent (Drake, 1994).

2 I 2 I 4

It would be expected that the bond energy in the X 2 molecules would decrease as the atoms become larger, Cl 2, Br 2 and I 2 shows the expected trend but F 2 does not fit the expected trend.

I.4 Radiochemistry of iodine Iodine has 33 radioactive isotopes (108 I 141 I) except 127 I which is a stable isotope with abundance of 100%. The produced 32 radioactive isotopes decay by EC, α, β+ and β. The radioisotopes of iodine with their nuclear characteristics and production nuclear reactions are listed in

Table (3) (Magill et al., 2006).

6

Table (3) Radioisotopes of iodine and their nuclear characteristics .

Isotope Halflife Decay mode Produced by

108 I 36 ms α 54 Fe( 58 Ni,x) 109 I 100 s P 58 Ni( 54 Fe,x), 92 Mo( 58 Ni,p2n) 110 I 0.65 s α, β+ 114 Cs α decay, 110 Xe EC decay, 111 I 2.5 s α, β+ 111 Xe EC decay 112 I 3.42 s α, β+ 58 Ni( 58 Ni,x), 112 Xe EC decay 113 I 5.9 s α, β+ 114 Cs ECP decay, 114 Ba EC α 114 I 2.1s β+ 115 Cs ECP 115 I 1.3 s EC Ag( 12 C,Xn), 92 Mo( 27 Al,2p2nγ) 116 I 2.9 s β+ 92 Mo( 28 Si,3pnγ), 106 Cd( 12 C,pnγ) 117 I 2.2 s β+ 114 Sn( 6Li,3nγ), 106 Pd( 16 O,p4n) 118 I 13.7 min β+ 118 Xe EC decay 119 I 19 min β+ 120 Cs ECP 120 I 53 min EC, β+ 114 Cd( 10 B,4nγ) 121 I 2.12 h EC, β+ 120 Te( 3He,d), 121 Sb( 3He,3nγ) 122 I 3.6 min β+ 122 Xe EC decay 123 I 13.2 h EC 122 Te(p,p), 122 Te( 3He,d) 124 I 4.15 d EC, β+ 124 Te(p,nγ), 125 Te(p,2nγ) 125 I 59.4 d EC 124 Te(p,p), 124 Te( α,t) 126 I 13.11 d β+, EC 126 Te(p,nγ), 127 I(n,2nγ) 127 I Stable(100% Abundance) 128 I 25 min β+,EC 127 I(n, γ), 127 I(d,p), 128 Te(p,nγ) 129 I 1.57X10 7y β 128 Te(p,p), 128 Te(3He,d), 130 Te( α,t) 130 I 12.36 h β, IT 129 I(n,γ), 130 Te(p,nγ) 131 I 8.02 d β 130 Te(p,p), 130 Te( 3He,d), 131 Te β decay

7

Continue of table (3) Isotope Halflife Decay mode Populating reactions

132 132 I 83.6 min β , IT Te β decay 133 I 20.8 h β, IT 134 Sb β n decay, 133 Te β decay 134 I 52 min β, IT 134 Te β decay 135 I 6.6 h β 135 Te β decay, 136 Xe(d, 3He) 136 I 84 s β 136 Te β decay 137 I 24.5 s β 137 Te β decay 138 I 6.4 s β 138 Te β decay, Fission 139 I 2.29 s β Fission 140 I 0.86 s β Fission 141 I 0.43 s β Fission

8

The major advantage of iodine isotopes is the type of radiation which emit on decay. Iodine isotopes in normal use are γemitters, their decay results in the emission of one or more photons whose energies are characteristic for the isotope. The second advantage of iodine isotopes is the relative shortness of their halflives. Since the maximum specific activity attainable with a given isotope is inversely related to its halflife, it is desirable to use an isotope with a short half life. The short halflives of iodine radioisotopes reduce the radiation exposure to patients treated with the radioactive isotope. Radioactive have proven to be useful for labeling both large and small molecules. The most frequently used radioisotopes of iodine in nuclear medicine are 125 I, 123 I and 131 I (Humm, 1986). 131 I decay by 100% β to give its stable daughter 131 Xe through the decay of 131mXe with the emission β particles at 606 KeV (89.4%) and the principle γ photons of 364 KeV (81.7%). Natural tellurium target used to produce 131 I via thermal neutron contains the following isotopic composition (Magill et al., 2006) as shown in Table (4). Table (4) Isotopes of natural tellurium and their abundance. Te 120 122 123 124 125 126 128 130 Abundance %) 0.09 2.55 0.89 4.74 7.07 18.84 31.74 34.08

In the production of I131 the activity contribution of Te121, Te121m, Te123m, Te124, Te125m, Te127, Te127m, Te129m, Te129, I129, Te131, Te131m are considered. The formation of Xe131, Xe 131m, Xe129 and Xe129m as a result of the decay of I131 and I129 are neglected (Mostafa, 2009).

9

I.5 Production of 131 I There are two types of nuclear reactions used for production of 131 I. a) By irradiating of natural or enriched tellurium130 compounds targets with thermal neutrons as shown in Fig. (1) (KAERI, 20002002).

(n, γ) 130 131m Te Te β (82%) 33.80% 30 h β(100%) 11.84 d 131 131m 131 I.T (18.%) I Xe Xe (stable) (n, γ) β (100%) 8.02 d I.T 130 Te 131 Te 25 min

Fig. (1): production of 131 I.

b) Extraction of iodine from fission products through (n.f) reactions and dissolution in acid or alkali media (Khalafi, 2005).

235 U (n,f) 131 I

Where the total fission yield for 131 I is 2.87%. Since the uranium fission technology requires a rather complicated and expensive facility, the tellurium irradiation would be the proper method for the isotope production laboratories established beside nuclear research reactors. The specific activity (i.e. radioactivity per gram of the target element) is a very important determinant factor in radionuclide production. The specific activity in millicuries per gram of the target element formed at any time during irradiation can be calculated by the following equation:

10

0.6 φ θ a S = (1 – e 0.693t/T) mCi/g (1) 3.7x10 7A

Where, S: the specific activity in mCi/g, φφφ: the thermal neutron flux in n.cm 2 .s 1, θθθ: the isotopic activation crosssection for thermal neutrons of the target isotope in barns (1 barn = 10 24 cm 2), a: the abundance of the corresponding isotope in the target material, A: the atomic weight of the target element, t&T are the irradiation time and the halflife of the product isotope, in the same unites, respectively.

I.6 Types of nuclear Reactions Reaction between an atomic nucleus (target) and another particle (projectile) is called nuclear reaction.

I.6.1 (n, γγγ) Reactions Most of the reactorproduced radioisotopes are products of the (n, γ) reaction. This reaction is also referred to as radioactive capture and is primarily a thermal neutron reaction. Here the product is an isotope of the target element itself and hence can't be chemically separated by conventional methods. Therefore, the specific activity is limited by the neutron flux available in the reactor (Choppin et al., 2002). Such types of 152 153 63 64 reactions include; Sm (n, γ) Sm (T1/2 = 46.75 h), Cu (n, γ) Cu (T 1/2 102 103 = 12.7 h) and Ru (n, γ) Ru (T 1/2 = 39.4 d). In some cases the (n, γ)

11 reaction produces a radioisotope which decays by e.g., β emission to a chemically different radioisotope: β X (n, γ) X1 Y

Where, X is the target material, X1 is the radioactive material of X and Y is the decay daughter of X1. Most radionuclide produced at "nocarrier added" (NCA) levels in reactors are produced via indirect reactions; such as 130 Te (n, γ,β)131 I (Grazman and Troutner, 1988).

(n, γ) β 130 131 131 Te Te I (T 1/2 = 8.02 d) 25 min

I.6.2 Multistage "double neutron" (2n,γγγ) Reactions For large crosssection, short halflives and long duration bombardment, secondorder (2n,γ) capture products may be formed. If the first order product is radioactive, then its concentration at any time is dependent on the decay constant, the crosssection for production of the secondorder product, as well as the crosssection for its own production. Example of radioisotopes produced by double neutron capture (2n,γ) 188 188 194 reactions, includes W (T 1/2 = 69 d; for Re) and Os (T1/2 = 6 y; for 194 Ir). Because of their long physical halflives and low production cross section, long irradiation periods are required for production of 188 W radioisotope, even in the highest neutron flux reactors.

12

I.6.3 (n, p) Reactions In some cases the absorption of neutron to emission of a proton as outgoing particle. Such a reaction is termed as (n, p) reaction and it is caused by fast neutrons having energy more than a particular value known as threshold energy. Hence, such a reaction is known as thershold nuclear reaction, as 47 Ti (n, p) 47 Sc at threshold energy 2.7MeV (Kolsky et al., 1998) and 67 Zn (n, p) 67 Cu at threshold energy up to 10MeV (Szelecsenyi et al., 1994).

I.6.4 (n, ααα) Reactions This reaction is also a threshold nuclear reaction as neutrons having energy above a specific value (threshold energy) are absorbed by the nucleus causing an alpha particle to be ejected. In some very special cases, the reaction is caused by thermal neutrons such as 6Li (n, α) 3H.

I.6.5 Fission Reactions Thermal neutron induced fission of uranium235 provides a host of (usually considered) carrierfree useful radioisotopes. Each fission provides two fission fragments. The fission products fall into two definite groups, one light group with a mass number of around 95 and a heavy group with a mass number of around 140. The fission yield of a is a fraction or the percentage of the total number of fission, which leads directly or indirectly to that nuclide. The total fission yield is 200%. In addition, some fission products undergo successive decays, leading to production of decay products forming a fission decay chain. Some of the most important fission products that find useful applications are; shortlived 131 I, 99 Mo and longlives 137 Cs and 90 Sr radionuclides (IAEA, 2005).

13

I.7 Definition of the labeled compound. A labeled compound has one of its atoms or larger structural units substituted in a way, which distinguishes that atom or unit from others. Labeled compounds of interest in the field of nuclear medicine will either be used in biochemical research or in medical diagnosis or therapy. Compounds used invivo for medical diagnosis is usually labeled with γ emitting isotopes to permit external radiation to the patient. Compounds labeled with β emitting isotopes such as 14 C, 3H, 35 S and 32 P are principally used in biochemical research.

I.8 Types of labeling. Two types of labeling are recognized: 1 Isotopic labeling: in which a compound is labeled with an isotope of an element already present in the compound, so that it is identical with the unlabeled compound. The majority of the labeled compounds are of this category. 2 Non isotopic labeling: in which compound is labeled with isotope of a foreign element not present in the unlabeled compound, for example labeling of proteins and peptides with radioactive iodine .

I.9 Methods of labeling pharmaceutical compounds. I.9.1 Chemical synthesis. In chemical synthesis, the complex molecules are prepared from simple and small isotopically labeled molecules. This synthesis may involve a single or multiplestep procedure and different physicochemical conditions determine the type and the yield of the labeled compound. The starting material used in the synthesis of organic compounds labeled with 14 C is

14

14 Ba CO 3 from which many compounds could be prepared. Chemical synthesis is normally the preferred one for the preparation of 32 P labeled compounds. Organic halides containing radioactive are prepared by halogenations under appropriate conditions of the various organic compounds using molecular halogen acids (Fonge et al., 2009).

I.9.2 Biosynthesis. Complex organic compounds are formed through biosynthesis of labeled compounds. In the 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 60 Co or 57 Co by adding the tracer to culture medium in which the organism Streptomycin Griseous is grown (Loch et al., 1991).

I.9.3 Recoil labeling In a nuclear reaction, when particles are emitted from a nucleus, recoil atoms or ions are produced which can form a stable bond which an organic or inorganic compound present in the target material. Several triturated compounds can be prepared in the reactor by the 6Li (n, α) 3H reaction. The compound to be labeled is mixed with a salt and irradiated in the reactor and 3H produced from the above reaction will then label the compound. The high energy of the recoil atoms makes the formation of stable chemical bonds difficult and results in poor yield, low specific activity and low radiochemical purity (Kim et al., 1994).

15

I.9.4 Excitation labeling This method uses radioactive daughter ions produced in nuclear decay processes for the labeling of various compounds of interest. 77 Kr decays to 77 Br and if the compound to be labeled is exposed to 77 Kr, 77 Br ions label the compound to form radiobrominated product. Also various proteins have been iodinated with 123 I by exposing them to 123 Xe which decays to 123 I.

I.9.5 Bifunctional chelates labeling Proteins may be radioiodinated through covalent bond formation between iodine and , usually in a tyrosine ring. The covalent bond formation is primarily a property of nonmetals. Many radionuclide of interest in nuclear medicine are metals that can be attached to proteins via complex or chelates formation. Most proteins do not possess metal binding, functional groups capable of forming stable bonds. Bifunctional chelates such as Ethylene DiamineTetra Acetic acid (EDTA) have been used in the labeling of various proteins. In this method, proteins are attached to a metal binding group and then labeled by chelation with a metallic radionuclide. The bifunctional chelate is a moiety which can be covalently, conjugated to proteins and which will form stable chelates for example 67 Galabeled desfsroxamine albumin (Hradilek and Kronlad, 1990).

I.9.6 Isotopic exchange reactions A large number of organic compounds have been labeled with radioactive isotopes by exchange method with minimum radiation hazards. In the exchange reaction one or more atoms in molecule exchange with a radioactive atom of the same element but of different mass under certain conditions ( ElShaboury, 2004).

16

AX* + BX BX* + AX

X has been exchanged with X* which is an isotope of the same element.

I.10 Radiopharmaceuticals. It is a radioactive compound intended to be introduced into the human organism for therapeutic or diagnostic purpose. The term radiopharmaceuticals cover a hundred of simple and complex radioactive chemicals used for invivo diagnostic or therapeutic applications ( 131 Irose bengal, 131 Iiodohippuric, 131 Idiiodone) and for invivo clinical diagnosis tests (Thomson et al., 2004). They are classified into five groups of preparation, based on the relevant nuclear chemistry operation: 1 Radioactive preparations obtained by irradiation of target followed by dissolution of this target. For example sodium24 as sodium bicarbonate, 42 as potassium carbonate and bromine82 as potassium bromide. 2 Radioactive preparations obtained by chemical separation of a radionuclide form an irradiated target. Such chemical separation may involve distillation, oxidation, reduction, adsorptiondesorption on an ionexchange resin or precipitation followed by redissolving. It is the most common method for the preparation of iodine131, 67, 51 and 32. 3 Radioactive preparations obtained by labeling and synthesis of organic molecules with radionuclides, examples of this method are

17

pharmaceutical compounds labeled with radioiodine which includes monoiodotyrosine, diiodotyrosine, triiodotyrosine, thyroxine and etc. 4 Radioactive colloidal preparation produced by precipitation of metal or salts such as colloid for example 198 and 90. 5 Isotope generators producing short lived radioactive daughters when required. 99 Mo/ 99mTc generator is the most widely used generator in nuclear medicine at the present time and in the near future.

I.11 Radionuclides used for diagnostic nuclear medicine. The radionuclide used for diagnostic nuclear medicine should have the following characteristics: 1 They must have short halflives from 10 seconds to 80 h; this is preferred because higher amounts can be given to the patients without significantly increasing the radiation doses. 2 They emit only γrays, no particles, this is an ideal situation (e.g. 99mTc) this would eliminate high local irradiation. 3 The energies of the emitted γrays should be in the range of 100300 KeV to be easily detected by present day gamma cameras. The main developed instrumentations used in nuclear medicine are: 1Single photon emission computed tomography 2Dual and triple head camera 3Positron emission tomography

I.11.1 Single photon emission computed tomography (SPECT) SPECT system I based upon a conventional γcamera detector mounted on an arm capable of 360˚ rotation around the patient. SPECT

18 capability adds 1520 % to the cost of basic γcamera / computer system. SPECT system allows a three dimensional visualization of radionuclide distribution. Its main application is the evaluation of brain and heart.

I.11.2 Dual and triple head camera It allows better evaluation of different organs with higher degree of resolution. Dual head allow examination of whole body interiority and poteriority within 15 minutes. Triple head is especially needed if the main field of application is neurology or cardiology.

I.11.3 Positron emission tomography (PET) PET technique relies on the coincidence counting in opposite detector of 511 KeV γphoton arising from positron annihilation. The major disadvantage of PET system is the cost and the need to a cyclotron in the same area for supplying shortlived positron emission radionuclides. Some commonly used γray emitting radionuclides and their routine production methods are summarized in Table (5).

19

Table (5): Routine methods of production of some commonly used γγγray emitters. Production data Mode of Main γγγray Nuclear Process energy range

Radionuclide T1/2 decay energy in (MeV) (%) KeV (%) 67 Ga 3.26 d EC(100) 93 (37) 68 Zn(p,2n) 26 → 18 185 (20) 99 Mo (generator) 2.75 d β(100) 181(6) 235 U(n,f) 740(12) 98 Mo(n, γ)

99mTc 6.0 h EC(100) 141(87)

111 In EC(100) 173(91) 25 → 18 2.8 d 247(94) 112 Cd(p,2n) 123 Te(p, n) 14.5 →10 123 124 I 13.2 h EC(100) 159(83) Te(p, 2n) 26 → 23 127 123 a I(p,n) Xe 65 → 45 124 123 Xe(p,pn) Xe 29 → 23 124 Xe(p,2n)123 Cs b 29 → 23 201 Tl 3.06 d EC(100) 6982 201 Tl(p,3n)201 Pb c 28 → 20 (Xrays) 166(10.2)

Note: a) 123 Xe decays by EC (87%) and β+ emission (13%) to 123 I b) 123 Cs decays by β+ emission and EC to 123 Xe. c) 201 Pb decays by EC (100%) to 201 Tl.

20

I.12 Preparation of radioiodinated compounds. The most common methods for the preparation of radioiodinated compounds can be classified as isotopic and nonisotopic exchange, electrophilic substitution and addition to double bonds. The obtained product differs from the original molecule and the radiopharmaceuticals are very unstable invivo. The most common reaction for introducing iodine into an organic compound is the iodination via electrophilic substitution. This method is applied to compounds containing substituted aromatic ring e.g. tyrosine ring. Labeling is achieved by introducing electrophilic iodine which is a foreign element to the compound to replace aryl hydrogen in the ring without any change in the properties of the compound. Such compounds will be foreign labeled (nonisotopically labeled compounds) (Seevers and Counsel, 1982). Almost all methods of radioiodination involve the use of oxidizing agents to convert iodide ion I to a positive oxidation state I + or the use of iodine in a partially electropositive oxidation state such as in ICL. Two schemes have been proposed for the iodination process (Berliner, 1966)

Scheme (A):

K1 + I2 + H 2O H 2OI + I

K2 + + H2OI + ArH ArHI + H 2O

K3 ArHI + ArI + H +

21

Scheme (B):

+ ArH + I 2 ArHI + I

ArHI + ArI + H +

+ For the iodination of aniline, it was shown that H 2OI is the possible substituting agent. It has been proved that the substitution agent + I2 or H 2OI may change with the substrate and the iodide ion concentration (Rayudu, 1983)

I.12.1 Electrophilic Substitution. I.12.1.1 ChloramineT (CAT) method The CAT method is the most widely used for radioiodination of proteins to obtain high specific activity. CAT is a mild oxidizing agent, it is the sodium salt of Nchloro4 toluenesulphonamide as given

+ H3C SO2NCl Na

The CAT method was discovered by (Greenwood and Hunter, 1963). When dissolving CAT in water, it decomposes to hypochlorous acid which in turn oxidizes radioactive iodide to active radioiodonium ion I+, species that is incorporated into the tyrosine residue of proteins with a high yield. The CAT method has an optimum labeling efficiency at

22 approximately pH 7 and the labeling efficiency is reduced at higher and lower pH values (Rayudu, 1983). The difficulty arising with the CAT method of radioiodination is that the compound to be labeled is exposed to powerful oxidizing conditions resulting in a number of undesirable side reactions such as chlorination (Baldwin and Lin, 1981), oxidation of thiol or thioester groups (Wood et al., 1980) and cleavage of peptide bonds (Alexander, 1994). In spite of these difficulties, CAT has been used to radioiodinate colchine via electrophilic substitution reaction, in which the reaction was quenched by the addition of sodium metabisulphite to reduce nonincorporated iodine before chromatographic analysis. the reaction parameters were studied to optimize the conditions for labeling and obtaining a high radiochemical yield (ElAzony et al., 2008). Solid phase version of chloramineT couple to polystyrene, called iodobeads has become available. In this method iodobeads are used to iodinate various peptides and proteins contains a tyrosine moiety. This technique has been claimed to be most successful with little denaturation of the protein and high labeling yield. Loch reported that a high efficiency preparation of 131 Iiodolisuride could be obtained using iodobeads (Loch et al., 1991).

I.12.1.2 Iodogen method Iodogen is 1, 3, 4, 6,tetrachloro3α, 6α–biphenyl glycouril, an oxidizing agent resembling 4fold chloramine–T as a promising agent for iodination of proteins. The main advantage of iodogen is its insolubility in water. It is dissolved in chloroform or methylene chloride and then evaporated on the inside walls of the reaction vial, thus permitting

23 labeling yield with very little contact of the organic compound with the oxidizing agent. The reaction is terminated by the transfer of the reaction mixture to a no coated tube. The advantage of this technique is that it is easy to perform, giving high labeling yield and causes less oxidative damage of the protein (Salisbury and Graham, 1981). The structure formula of iodogen is as follow

O

Cl Cl N N

N N Cl Cl

O

I.12.1.3 Enzymatic method In Enzymatic iodination, enzymes such as lactoperoxidase and chloroperoxidase and very small amount of hydrogen peroxide added to the iodination mixture containing radioiodine and the compound to be labeled. The enzyme is used to catalyze the oxidation of iodide in the presence of small amounts of H 2O2. This method is the mildest and the most useful technique which was used for the iodination of many proteins and hormones used in radioimmunoassay (Bockisch et al., 1993). Denaturation of proteins or alteration in organic molecules is minimized. Enzymatic method has been reported to be a useful technique for radioiodination of several compounds such as tyrosine, uracil, histidine and monoclonal antibodies (Hadi et al., 1977). Enzymatic radioiodination

24 is a milder alternative technique that uses chemical oxidants especially when the source of hydrogen peroxide is another enzyme system such as glucose oxidase (Morrison, 1990). This method has the disadvantage of long reaction time.

I.12.1.4 Electrolytic method Many amino acids, proteins and hormones can be radioiodnated by this mild method, which consists of the electrolysis of a mixture of radioiodide and the compound to be labeled in saline solution. Electrolysis releases reactive iodine that labeled the compound. The reactive iodide that was produced by the reaction can be reoxidized. Slow and steady liberation of iodine causes uniform iodination of the compound and a high labeling yield can be achieved. This method has the advantage of not exposing the material to powerful oxidizing conditions but requires some special equipment. Compounds radioiodinated by this method are estradiol and their derivatives (Moore and Wolf, 1978).

I.12.2 Nucleophilic substitution. Another method for radioiodination of organic compounds is the exchange technique where the inactive iodine in the iodoorganic compounds was replaced by the radioactive iodine as shown in the exchange of radioiodide with orthoiodohippuric acid (Khater et al., 2006). The reaction is preceded by nucleophilic substitution (IPSO substitution) of iodine atom via an intermediate in which both the inactive and radioactive atoms are symmetrically bond to the carbon atom. The

25 rate of the reaction is given by the rapture of the CI bond, which is dependent on the temperature. Isotopic exchange reactions often require an activation energy, which can be provided by thermal agitation. Exchange reactions can be accelerated by the action of light or ionizing radiation. Since most of the iodine atoms in the final product will be 127 I, it is not possible to prepare compounds with high specific activity by this method.

I.12.2.1 Exchange in solvent The last complex method of radioiodination of small organic molecules is the substitution of radioiodine of a stable iodine atom already incorporated in the molecule. This could be affected by heating isotopic or exchange medium may be water, buffer solution adjusted to a favorable pH or organic solvent such as ethanol or acetone. The solvent must be able to dissolve both the organic molecule and the inorganic iodide. Many organic molecules have been radioiodinated by this method e.g. orthoiodohippuric acid and fatty acids (Hradilek and Kronlad, 1990).

I.12.2.2 Exchange in melt. Compounds which do not exchange well with iodide in solvents can give better yields when the iodocompounds is melted with radioiodide. The simplest form is the compound itself, which must be stable at its melting point and has a high dielectric constant to dissolve the radioiodide. This technique was first developed for the exchange labeling of miodohippuric acid (Elias et al., 1973) and was used to label

26 cholesteryl4iodobenzoate and 4iodophenyl alanine (Lambrecht et al., 1974). To overcome the difficulties with low dielectric constant compounds, the exchange is done in a melt of acetamide at about 180 ◦C (Elias and Lotterhos, 1976). Acetamide melts at about 82 ◦C and is stable above 200 ◦C. Another type of melt was done by heating the substrate with radioactive iodide in ammonium sulfate at (120160 ◦C). The author suggested that this causes a gradual increase in the acidity of the exchange medium via in situ decomposition of ammonium sulfate with loss of ammonia. Farah et al. studied the labeling of metaiodobenzylguanidine (mIBG) by isotopic exchange in melt (Farah et al., 1999). ElWetery et al applied the exchange in melt technique in the labeling of 16Br hexadecanoic acid with radioactive iodide using ammonium acetate as a catalyst (ElWetery et al., 1997).

I.12.2.3. Exchange for bromine Labeling by radioiodine can be performed via iodine for bromine exchange resulting in high specific activities provided the radioiodinated product can be separated efficiently from the brominated compound. Many radiopharmaceuticals have been prepared by this method both in solvents and in melt such as long chain fatty acids which is used in heart imaging. This method was used for the preparation of large quantities of nocarrieradded (NCA) 17–123 I iodoheptadecanoic acid (Argentini et al., 1981).

27

I.12.2.4 Catalyzed exchange. In isotopic exchange reactions, catalysts are used to improve the radiochemical yield and decrease the reaction time especially in radioiodination reactions with shortlived 123 I isotope. The presence of dicyclohexyl18crown ether6 catalyzed the exchange of sodium iodide with alkyl bromide (Liu et al., 1981). metal or copper (I) salts have been reported to catalyze the exchange of iodoaromatics with radioiodine in high radiochemical yields. Such results have been obtained in the exchange of mIBG (Verbruggen, 1987), and 15(4iodophenyl) 9 methyl pentadecanoic acid (Mertens et al., 1986). Exchange between radioiodide and hydrogen to form 4iodoantipyrine has been catalyzed by silica gel (Boothe et al., 1992). Copper catalyzed radioiodination of 3 iodotyrosine and 4iodophenyl alanine were prepared by this method with relatively high radiochemical yield and purity (Farah and Farouk, 1997).

I.13 Purification of radioiodinated compounds. Many radioiodination techniques are used in the preparation of iodine labeled compounds. Unreacted iodide (radiochemical impurity) must be removed from the solution. For microscale preparation, paper or thin layer chromatography can be applied. For largescale production, when high activities are used, column chromatography is used for purification of iodocompounds from unreacted radioiodine (Anghileri, 1974). Anion exchange resins Dowex1, Dowex2 and Amberlite IR400 are the most widely used. Free iodine and inorganic iodide are adsorbed

28 by the resin. Optimum separation is achieved by choosing the proper eluent. High performance liquid chromatography (HPLC) has been developed as an efficient tool for the purification of radioiodinated compounds (Meyer, 1982). Different materials are used for packing the columns such as Lischrosorb, Silica Gel, Octadecyl silane and Dextrane. The iodinated compound is separated from the reaction mixture on HPLC column according to its retention time by a solvent HPLC grade. This separation is monitored by detectors such as UV detectors and γdetectors. According to the signals of the detector, the eluted fractions of the desired iodinated compounds can be collected by a fraction collector, evaporated and the residue dissolved in an isotonic silane. The product is sterilized using a bacteria filer and the radiopharmaceutical is ready for use in nuclear medicine application.

I.14 Quality control. A radioisotope used in nuclear medicine must be controlled for the presence of chemical, radiochemical and radionuclidic components (impurities) other than those intended to be present. Impurities may arise during preparation and storage of radiopharmaceuticals. These impurities will frequently modify organ distribution and specificity, possibly leading to an incorrect diagnosis of the patient health. In evaluating the quality of a radioisotope, several properties such as radioactivity calibration, radionuclidic, radiochemical and chemical purity of the product radioisotope must be noted to assure its safety and efficiency. Sterility and apyrogenicity are absolute quality requirements for radiopharmaceuticals intended for potential administration. Since radiopharmaceuticals are

29 radioactive compounds used for diagnosis and therapeutic treatments of human diseases, a quality control tests are carried out to insure the purity, safety and efficiency of these products and the suitability for the intended use in nuclear medicine application (Kenneth and Jansholt, 1977). Radioisotope production and isolation. Impurities in radiopharmaceutical synthesis. Synthesis sidereactions leading to labeled derivatives. Incomplete preparative separation. Breakdown during storage.

I.14.1 Chemical purity. Chemical purity is the determination of the stable impurities. It is primary determined by the quality of the different chemicals used. The importance of measuring the chemical purity is related to their effect where, they change the chemical and physical properties of the desired radioisotope. Spectrographic analysis and colorimetric spot tests are the two main methods used for measuring the chemical purity. The raw materials used for the preparation of the radiopharmaceuticals are tested by different analytical techniques to ensure their suitability.

I.14.2 Radionuclidic purity . Radionuclidic purity refers to the fraction of the total radioactivity that is present as the specified radioisotope. Foreign radionuclides originate from the target material impurities and/or side nuclear reactions during irradiation and can not always be removed by chemical purification methods. The radionuclidic purity becomes so important with

30 the passage of time since, a minor longlived radioisotope may become the predominate one present. Radionuclidic purity is checked by the γ spectrometer and the decay curve of the desired radionuclide. The radionuclidic purity of 131 I through a 364 KeV γline, while Te was followed through a 159 KeV γline emitted by 123 Te (119.7 d). This isotope is selected among the other isotopes due to its long halflife and because it did not decay to 131 I.

I.14.3 Radiochemical purity. The radiochemical purity is the proportion of the activity in the specified chemical form. Radioiodine may exist as molecular iodine, iodate, periodate and iodoorganic compound. In the labeling of organic compounds, the unreacted iodide represents a radiochemical impurity and must be removed by the purification methods mentioned before, namely TLC, HPLC and electrophoresis. Non regiospecific iodination method leads to a mixture of isomers in electrophilic substitution of aromatic compounds. Efficient and rapid chromatographic techniques like HPLC can perform the separation of isomers . The chromatographic separation of radioiodine species is shown in Table (6).

31

Table (6) Chromatographic separation of radioiodine species (Baldwin, 1986)

Stationary phase Mobile phase Rf values 1 IO 3 IO 4

1paper Whatmann no.1 70% methanol 0.7 0.3 0.3

Whatmann no.1 nbutanol saturated with 3N 0.31 0 0 ammonia 2TLC silica gel Isopropyl alcohol: Ethyl acetate: 6 M ammonia: acetone 0.8 0.13 (35:30:25:20) 3HPLC RP18 10% acetonitrile in 0.05 M phosphate 1.2 0 5.36

buffer (2mMBu 4NH 4OH)

I.15 Apyrogenicity, Toxicity and Biological distribution Radioiodinated compounds used in nuclear medicine for diagnosis and therapeutic treatments of human diseases must be sterile and pyrogen free. Apyrogenicity is tested in rabbits. The toxicity and biological distribution are preformed in mice, according to British Pharmacopoeia , (2009).

32

Experimental

II.1 Materials All chemicals and reagents used were from commercial sources and analytical grade.

Tellurium dioxide (TeO 2, M.W=159) from Merck Chemical Company. Iodogen (1, 3, 4, 6tetrachloro3ααα6αααbiphenyl glycoluril, M.W.432.09) from Pierce Chemical Company. ChloramineT [Nchloroptoluene sulfonamide sodium salt (ChT)] M.W.227.65 from Aldrich Chemical Company.

Hydrogen peroxide, H 2O2 (30% in acetic acid) from Fischer Scientific Company. Sodium hydroxide (pellets) (NaOH, M.W=40) from Fluka Chemical Company. 4[2hydroxy3(isopentylamino) propoxy] indole (Yindole), M.W. 275 and [(1ethyl2pyrrolidyl) methyl]2,3dimethoxy5(tributylstannyl) benzamide (Epidepride), M.W. 541 were kind gift from Edmonton Radiopharmaceutical Centre (ERC), Edmonton. Canada. Solvents for highpressure liquid chromatography are high purity grade and degased before use.

II.2 Equipment Spectrophotometer: Shimadzu Model UV/VIS 160A Japan . γ Spectrometer : Multichannel analyzer EG& ORTEC, Model GEM18190 with High purity germanium (HPGe) coaxial detector (USA). Crystal

33 diameter54.2mm, crystal length 42.0mm and end cap to crystal 3mm;high voltage 1500V. HPLC: MerckHitachi Model consists of L6000 pump, L4000 UV/ VIS spectrophotometer Lichrosorb, RP18 column (250x4 mm), Japan. γScintillation counter: Scaler ratemeter SR7 type fitted with well type NaI (TI) crystal detector. pH meter: Bench type, Hanna, H18418 with microprocessor electrode, pH ranging 014. Analytical balance: Bosch S 2000 ranging from 0.5mg to 200g. Tight glass jar: for chromatography (20cm length and 2cm diameter). Thin layer chromatography (TLC): aluminum sheets (20x25cm) SG60 F254 Merck . Apparatus for separation of iodine131 The apparatus used for dry distillation method is shown in Figure (2). The distillation quartz tube (ө = 4.5 cm, length =16.5 cm) is placed in an oven (digital oven maximum temperature is 900 ◦C). It connected from one side to a raw of taps (one containing 5M H 2SO 4 acid solution then a condenser ended with 20 ml vial containing 5 ml 0.1M NaOH solution). A vacuum pump allows air to flow through the distillation tube. The temperature is raised gradually for 100 to 700 ◦C via temperature scale of 100 ◦C / 5 min and rest at this temperature for the required time. The air 131 steam will carry the I through H2SO 4 solution, which selectively absorbs only any tellurium components. Then, the air flow will carry the 131 I vapors to the condenser where it will be collected via passing through 0.1M NaOH solution.

34

5M H 2SO 4

Figure (2) Schematic diagram of the distillation apparatus used for 131 production of I from irradiated TeO 2 target.

35

II.3 Preparation and irradiation of the target

A known weight (5g) of natural TeO 2 as target material was put carefully in an aluminum irradiation can to preserve the target material from any contamination through the reactor cooling circuit. The aluminum can was introduced into another outer leak proof aluminum. The target was then irradiated for 12 h in the vertical channel of 22MW watercooled research reactor ETRR2 at Inshas. The average thermal neutron flux density was 1x10 14 n/cm 2 sec; the irradiated target was transported from the reactor to the laboratory. The irradiated target was left to cool for at least 48 h before distillation to give the chance for the shortlived radioisotopes to decay.

II.4 Factor affecting the production of 131 I II.4.1 Effect of oven temperature on the releasing 131 I activity The effect of temperature on the production of 131 I. The irradiated

TeO 2 target material was placed in the furnace which was heated rapidly and successively to increase temperature of 500 ◦C and maintained for 20 min. The process is repeated for other targets at temperatures of 550, 600, 650 and 700 ◦C, respectively. The produced radioiodine was trapped through a condenser containing 0.1 M NaOH solution. The released radioiodine was measured using multichannel analyzer; also, the tellurium concentration was determined in the separated radioiodine spectrophotometrically (Beyer and Gonzales, 2000).

36

II.4.2 Effect of distillation time on the releasing 131 I activity and the

concentration of tellurium (IV) as Na 2TeO 3 in the separated radioiodine The effect of distillation time on the production of 131 I and on the tellurium concentration in the separated radioiodine was studied at different time intervals (10, 20, 30, 40, 50, 60 min) keeping the temperature constant at 700 ◦C. The produced radioiodine was trapped through a condenser containing 0.1 M NaOH solution. The released radioiodine was measured using multichannel analyzer the tellurium concentration was determined in the separated radioiodine spectrophotometrically .

II.4.3 Sodium hydroxide concentration The effect of sodium hydroxide concentration for sorbing the produced radioiodine was studied keeping the temperature 700 ◦C and the distillation time 30 min. The produced 131 I vapor was collected through sorption into different concentration of sodium hydroxide (0.01, 0.05, 0.1, 0.2, 0.5, 0.8, 1.0M). The collected 131 I in each concentration was radiometarically analyzed using a multichannel analyzer.

II.5 Quality control on the released 131 I II.5.1 Chemical purity Tellurium was measured and determined spectrophotometrically.

Standard calibration curve of tellurium ion Figure (3) shows the standard calibration curve between tellurium concentration and the absorbance. The tellurium dioxide solvated in conc. Hydrochloric acid and small portion of conc. Nitric acid of concentration

37 range of 0.740 g Te/ml. A certain volume of the standard solution of tellurium was diluted with the proper volume of water to obtain the desired concentration. The absorbance of each sample was measured as follow:

1) 30 ml of 2M HCI was added to acidify one ml of the sample; 5ml of poly vinyl alcohol (2%) was then added followed by 5ml of the 20% stannous chloride. A brown color was observed depending on the concentration of tellurium. The solution was completed to 50 ml volume by water (Marczenko, 1986).

2) A blank solution was prepared by adding all the previous components of the same concentrations and volumes except Te solution.

3) The absorbance of each sample was measured on the Spectrophotometer at a wavelength of 320 nm, after correction of the absorption due to other components using the blank solution.

4) A calibration curve was drawn between the concentration of Te in g/ml and the corresponding absorbance.

5) The Te solution of unknown concentration was treated as previously mentioned, its absorbance was measured and its concentration was derived by the aid of the standard calibration curve.

38

0.8

0.6

0.4

0.2 UV Absorbance, arbitrary units

0.0 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 Concentration of Te( g/ml)

Fig.(3): Standard calibration curve of tellurium.

II.5.2 Radionuclidic purity To identify the presence and contribution of foreign radionuclide impurities in the 131 I produced from tellurium by dry distillation method, the released radioiodine was counted using HPGe detector to follow the radioactive isotopes. Alternatively, to determine the percentage of the produced 131 I the gross γ radioactivity is immediately measured using NaI (TI) scintillation counter and at predetermined different time intervals. The corresponding decay curve is plotted and the contribution of the longlived radioisotopes in the produced 131 I obtained from the tail of the decay curve.

II.5.3 Radiochemical purity For determination of the 131 I radiochemical purity, TLC technique is used where, a drop of the produced radioiodine containing

39 radioactivity~ 5X10 3 cpm was spotted on a Whatman filter paper No. 1 strip (30X2 cm) and dried. The strip was then suspended into the chromatographic jar containing a solvent mixture of 70% methanol when the solvent front had mounted 10 cm; the paper strip was taken out, dried and cut into pieces 0.5 cm in width. The cpm in each was counted (Sheh et al.,

2000) as a relative front R f.

Distance (cm) from the start line to the radioactivity peak position Rf = ______Distance (cm) moved by the solvent from the start line to the solution front

II.6 Iodination of 4[2hydroxy3(isopentylamino) propoxy] indole (Yindole) The iodination processes were achieved using two oxidizing agents, chloramineT and iodogen. The structure formula of Yindole can be represented as follows: H

N

OH H O N C2H5

C H 2 5 II.6.1 Cold iodination of Yindole II.6.1.1 ChloramineT method To a reaction vial, 100 l methanol containing 1mM Y indole were added, followed by 100 l phosphate buffer (0.1M, pH 7) containing 1mM KI. The reaction was started by addition of 100l of freshly prepared

40 phosphate buffer (0.1M, pH 7) containing 1mM of chloramineT. The reaction was allowed to proceed for suitable time after which the reaction was quenched by the addition of 50l phosphate buffer (0.1M, pH 7) containing 15.8 mM of sodium metabisulphite to reduce nonincorporated iodine before chromatographic analysis. Each component in the reaction mixture was injected onto RP18 HPLC column and eluted after a definite retention time (R t) by a mixture of acetonitrile: 0.1 M sodium bicarbonate (1:1) at a flow rate of 1ml / min and the reaction mixture was also injected onto RP18 HPLC column for analysis.

II.6.1.2 Iodogen method Iodogen is insoluble in water, so it can be used as a thin film plated onto the bottom of the glass vial which the reaction is performed. 20l of methanol containing 1mM Y indole were added to iodogen coated tube; followed by addition of 100 l phosphate buffer pH 7 containing 1mM KI. The reaction mixture was allowed to proceed for 30 min. The reaction mixture was removed to another vial to stop the reaction. 10 l of the reaction mixture was injected onto RP18 HPLC column for analysis.

II.6.2 Radioiodination of Yindole. II.6.2.1 ChloramineT method To a reaction vial, 100 l methanol containing 1mM Y indole were added, followed by addition of 100 l (500Ci) of Na 131 I. The reaction was started by addition of 100l of freshly prepared phosphate buffer (0.1M, pH 7) containing 1mM of chloramineT. The reaction was allowed to proceed

41 for 30 min then which the reaction was quenched by the addition of 50l phosphate buffer (0.1M, pH 7) containing 15.8 mM of sodium metabisulphite to reduce nonincorporated iodine before chromatographic analysis. 10 l of the reaction mixture was injected onto RP18 HPLC column for analysis.

II.6.2.2 Iodogen method Iodogen is insoluble in water. It can be used as a thin film plated onto the bottom of the glass vial which the reaction is performed. 20l of methanol containing 1mM Y indole were added to iodogen coated tube; followed by addition of 100 l (500Ci) of Na 131 I. The reaction mixture was allowed to proceed for 30 min then the reaction mixture was removed to another vial to stop the reaction. 10 l of the reaction mixture was injected onto RP18 HPLC column for analysis. The factors affecting the radiochemical yield of Yindole using chloramineT and iodogen as oxidizing agents were investigated such as Yindole concentration, pH of the medium, reaction time and oxidizing agent concentrations.

II.7 Iodination of [(1ethyl2pyrrolidyl) methyl]2,3dimethoxy5 (tributylstannyl)6 hydroxy benzamide (Epidepride) The iodination processes were achieved using two oxidizing agents, chloramineT and hydrogen peroxide. The structure formula of Epidepride is as follows:

42

H N CONHCH2 C H HO OMe 2 5

R OMe

R= tributyl

II.7.1 Cold iodination of Epidepride II.7.1.1 ChloramineT method To a reaction vial, 5l ethanol containing 3.69 mM epidepride, 50 l phosphate buffer (pH 3.4) containing 1mM KI, 5l of (10N) HCl. The reaction was started by addition of 10l of freshly prepared phosphate buffer (pH 3.4) containing 50nM of chloramineT. The reaction was allowed to proceed for 20 min after which the reaction was quenched by the addition of 50l phosphate buffer (pH 3.4) containing 15.8 mM of sodium metabisulphite to reduce nonincorporated iodine before chromatographic analysis. The reaction mixture was neutralized with 10 l of (4N) ammonium hydroxide. Each component in the reaction mixture was injected onto RP18 HPLC column and eluted after a definite retention time (R t) by a mixture of (0.1M, pH 7.5) phosphate buffer : ethanol (30:70) at a flow rate of 1ml/min and the reaction mixture was also injected onto RP18 HPLC column for analysis.

43

II.7.1.2 Hydrogen peroxide method To a reaction vial, 5l ethanol containing 3.69 mM epidepride, 50 l phosphate buffer (pH 3.4) containing 1mM KI, 5l of (10N) HCl. The reaction was started by addition of 10l of 3% H 2O2. The reaction was allowed to proceed for 20 min after which the reaction was quenched by the addition of 50l phosphate buffer (pH 3.4) containing 15.8 mM of sodium metabisulphite to reduce nonincorporated iodine before chromatographic analysis. The reaction mixture was neutralized with 10 l of (4N) ammonium hydroxide. 10 l of the reaction mixture was injected onto RP18 HPLC column for analysis.

II.7.2 Radioiodination of epidepride. II.7.2.1 ChloramineT method To a reaction vial, 5l ethanol containing 3.69 mM epidepride, 50 l Na 131 I (500Ci ) were added, 5l of (10N) HCl. The reaction was started by addition of 10l of freshly prepared phosphate buffer (pH 3.4) containing 50nM of chloramineT. The reaction was allowed to proceed for 20 min after which the reaction was quenched by the addition of 50l phosphate buffer (pH 3.4) containing 15.8 mM of sodium metabisulphite to reduce nonincorporated iodine before chromatographic analysis. The reaction mixture was neutralized with 10 l of (4N) ammonium hydroxide. 10 l of the reaction mixture was injected onto RP18 HPLC column for analysis.

II.7.2.2 Hydrogen peroxide method To a reaction vial, 5l ethanol containing 3.69 mM epidepride, 50 l of Na 131 I (500Ci), 5l of (10N) HCl . The reaction was started by addition

44 of 10l of 3% H 2O2. The reaction was allowed to proceed for 20 min after which the reaction was quenched by the addition of 50l phosphate buffer (pH 3.4) containing 15.8 mM of sodium metabisulphite to reduce non incorporated iodine before chromatographic analysis. The reaction mixture was neutralized with 10 l of (4N) ammonium hydroxide. 10 l of the reaction mixture was injected onto RP18 HPLC column for analysis. The factors affecting the radiochemical yield of epidepride using chloramineT and hydrogen peroxide as oxidizing agents were investigated such as epidepride concentration, pH of the medium, reaction time and oxidizing agent concentrations.

II.8 Biological distribution of the labeled compound in mice The biodistribution studies were carried out by injection of 131 IY indole in the tail vein of groups of mice, (each group of three mice) and then take part of organ of each mouse to activity counting system.

45

Results and Discussion

III.1 Irradiation of target material 131 I is produced from natural tellurium dioxide target, irradiated through thermal neutrons by the 130 Te (n, γ) 131 Te decays by β to 131 I and can be separated by dry thermo distillation (IAEA, 2005). On irradiating 5 g target of natural tellurium dioxide in reactor of thermal neutron flux of 1x10 14 n.cm 2.s 1, the calculated specific activity of 131 I using equation (1) at different irradiation time intervals. The specific activity of the produced 131 I after irradiation is high and meets the requirements for the production of small scale of 131 I (Fatima et al., 2002). Figure (4) shows a typical γ ray spectrum of the targets irradiated for 12 h in the ETRR2 reactor at a thermal neutron flux of 1X10 14 n.cm. 2.s 1 after cooling time of 48 h. From the figure it is found six peaks for 131 I at 84, 285.2, 325.1, 364, 636 and 723 KeV and the productiondecay reactions of 131 I as shown in Figure (1). The irradiated targets contain in addition to 131 I, the radioactive isotopes of tellurium. The production and the ratios of decay branches were cited from Nuclear Decay Data as follow (Nuclear Decay Data, 2006)

(n, γ) EC (11.6 %) 120 121m 121 Te Te (154 d) Sb (stable) (0.09%) (88.4 %) I.T E.C (100%)

121 Te (16.8 d)

46

The γray spectrum contains 123mTe (157 KeV) the production and decay equation as follow:

(n, γ) I.T (100%) EC (100%) 122 123m 123 123 Te Te (119.7 d) Te (1.24 x10 13 y) Sb (stable) (2.55 %)

(n, γ) I.T (100%) 124 125m 125 Te Te (57.4 d ) Te (stable) (4.74%)

124 Te is present in the target material (4.74%) that produce 125mTe which has relatively long T 1/2 and appeared clearly in Figure (10).

(n, γ) β (2.4%) 126 Te 127mTe (109 d) 127 I (stable) (18.84%)

I.T (97.6%)

β (100%) 127 Te (9.35 h)

(n, γ) β(37%) β (100%) 128 129m 129 129 Te Te ( 33.6d) I ( 1.57x10 7 y) Xe (31.71%)

I.T (63%)

β (100%) 129 Te (69.6m)

47

) )

Te(24.9KeV I (723KeV) 121

Te (150, 157KeV) 131

m

Te, I ( 364I KeV) m 123

Te,

131

127 131

Te, ,

131 I (84I KeV) KeV) KeV I (285.2KeV) 121Te

131

Te (102Te KeV) 131

Te (458KeV) Te (211.3KeV) Te, 694

131 636.7 I (325.1KeV) ( 121m I 129m Te Te (417KeV) Te,

131 131m

129 Te(507 KeV) Te(572 KeV)

127 121 131

121

Counts per channel, arbitrary units units arbitrary channel, per Counts

129

γ ray energy, KeV

Fig. (4) γ ray spectrum of thermal neutrons irradiated TeO 2 target

material measured after 48 h from the end of irradiation

48

III.2 Dry distillation method III.2.1 Effect of oven temperature on the releasing 131 I activity A figure (5) show that the optimum oven temperature required to release the maximum 131 I activity (103 mCi) is 700 ◦C. The removal of radioiodine was initially low at low temperatures (500, 550, 600 and 650 ◦C). The concentration of tellurium in the released radioiodine was very small and not detected spectrophotometrically. The experiment did not exceed the temperature of 700 ◦C in order to avoid reaching the ◦ melting point of TeO 2 (733 C).

III.2.2 Effect of distillation time on the releasing 131 I activity and on the tellurium (IV) concentration in the separated radioiodine Figure (6) shows the effect of distillation time at different temperatures and the activity of iodine131 released which confirms that the activity of 131 I was initially low but, as soon as a distillation time of 30 min. at oven temperature of 700 ◦C under these conditions the radioiodine activity is almost maximum. Whereas, at oven temperature of 650 ◦C the optimum distillation time is 50 minutes. At oven temperatures of 600, 550 and 500 ◦C, a distillation time of 60 minutes is still not enough to release the maximum radioiodine activity. At oven temperature of 700 ◦C the tellurium concentration in the released radioiodine is measured using UVspectrophotometer. The results are obtained at optimum temperature 700 ◦C Table (7) which proves that the tellurium concentration in the released radioiodine was not detected after the first 20 min., tellurium concentration becomes 0.8 g/ml after 30 min. The tellurium concentration in the released radioiodine is below the permitted

49 limit for medical application (Debondt, 2001). At 40, 50 and 60 min, tellurium concentrations were 15, 21 and 28 g/ml, respectively. These tellurium concentrations are above the permitted limit for medical application. This proves that the tellurium loss from target was not detected extremely very small and may be deposited in the inner walls of the quartz tube and/or sorbed through 5M H 2SO 4 solution; only an infinitesimal fraction reaches the produced radioiodine (Alanis and Navarrete, 2001). The results obtained from Figure (6) so; it is recommended an oven temperature of 700 ◦C and distillation time of 30 minutes.

50

100

80 Activity,mCi 60

40 500 600 700 800 Oven temperature, C

Fig. (5): The effect of oven temperature releasing 131 I activity.

120

700 0 C

100 600 0 C 650 0 C 550 0 C

500 0 C 80

60 Activity, mCi

40

20

0 0 10 20 30 40 50 60 70 Time, min Figure (6): The effect of time and oven temperature on the released 131 I activity.

51

Table (7): The concentration of tellurium in the separated radioiodine at optimum temperature 700 ◦C Distillation time (min) Tellurium concentration (g/ml) 20 Not detected 30 0.8 40 15 50 21 60 28

III.2.3 Effect of sodium hydroxide concentration The released radioiodine vapors are collected using NaOH. Figure (7) shows the effect of NaOH concentration on collection of the produced radioiodine via distillation technique using the above optimum conditions (temperature 700 ◦C and distillation time 30 min) Different concentrations of NaOH (0.001, 0.05, 0.1, 0.15, 0.2, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50 M) were prepared. The sorption curve is more or less independent on the NaOH concentration with the maximum value of 103 mCi at 0.1M. Below this concentration, about 80 % is sorbet .

III.3 Quality control of the produced 131 I III.3.1Chemical purity 131 The concentration of tellurium in the produced I was determined spectrophotometrically. It was found that under the determined conditions, the concentration of tellurium was below the permitted limit for medical purpose (0.8 g/ml), there are no necessary to introduce a

52 chemical and physical treatment to improve chemical purity and to reduce the tellurium contamination level in the produced 131 I (Debondt, 2001).

III.3.2 Radionuclidic purity Radionuclidic purity of produced radioiodine was measured using multichannel analyzer. Figure (8) shows the γ ray spectrum of the produced 131 I. Figure (8) shows the presence of only 131 I radionuclide. The Radionuclidic purity was also measured by using the decay curve measurements over time span of 0 to 56 days. Figure (9) shows the decay curve of the produced 131 I via dry distillation technique. The decay curve indicates that the 131 I produced was of 99.9 %.radionuclidic purity.

53

120

100

80 , , mCi

60 activity

Produced 40

20

0 0.001 0.051 0.101 0.151 0.201 0.251 0.301 0.351 0.401 0.451 0.501

NaOH concentration, M

Fig.(7): Effect of NaOH concentration on sorption of the produced 131 I.

54

I (364.3 I KeV)

131

I(284.6 KeV) I(80.6 KeV) I637.1KeV 131 131 I (723 .8KeV)

131 131

I(503.1Kev) Counts perchannel, arbitraryunits

131

Gamma ray energy, KeV

Fig.3. Gammaray spectra of 131 I product solution Fig.(8): γ ray spectrum of the produced 131 I.

x 10 3 10

half activity

1

impurities

Activity, cpm Activity, 0.1

half life of 131 I (8.02 d) 0.01 0 5 10 15 20 25 30 35 40 45 50 55 60 65 Time, days

Fig.(9) Decay curve of the produced I131.

55

III.3.3 Radiochemical purity Figure (10) shows the radiochromatogram of produced 131 I , thin layer chromatography using 70% methanol as developing solvent for separation of radioiodide. The solvent separates radioiodide at R f 0.7 and the other radiochemical impurities (iodate and periodate) at R f 0.3. The corresponding radiochemical purity was found ≥ 98.7%. This result was identified by γspectrometric analysis where the radioiodide is concentrated at Rf ~ 0.7 in an agreement with the chemical form of iodide (Beyer and Gonzales, 2000).

III.4 Optimization for the production of Iodine 131 All the above investigated data are summarized in Table (8) Figure (11) shows the 131 I production flow sheet via dry distillation method. The results obtained prove that 131 I can be produced through dry distillation. Table (8) Summary of 131 I production via dry distillation technique.

TeO 2 target weight 5 g Reactor neutron flux 10 14 n/cm 2.s

Irradiation time 12 hours

Cooling time 48 hours

Oven temperature 700 0C

Distillation time 30 minutes

Produced 131 I activity 103 mCi

Radionuclidic purity 99.9%,

Radiochemical purity ≥98.7%, in the iodide form Chemical purity ∼ 0.8 g/ml of Te.

56

x 10 3 6

5 Rf= 0.7

4

3

2 Radioactivity, cpm Radioactivity,

1

0 0 1 2 3 4 5 6 7 8 91011 Distance, cm

Fig. (10) Radiochromatogram of the produced 131 I.

57

5 g TeO 2

Irradiated at thermal neutron flux of 10 14 n/cm 2.s for 12 h

131 mTe, 131 Te, and 131 I (Irradiated TeO 2 target)

Dry distillation at 700 ◦C Separated 131 I for 30 min vapors 5M H 2SO 4 Condenser

5 ml 0.1M NaOH

131 Quality control Produced I

Chemical Radionuclidic Radiochemical

Fig.(11) Schematic flow sheet of the 131 I production process via dry distillation technique.

58

III.5 Radioiodination of Yindole III.5.1 introduction High affinity radiolabeled antagonist have proven very useful for the direct assay of βadrenoceptors in membrane preparation from variety of tissue sources (Williams and Lefkowitz, 1979). 3Hdihydroalprenol (3H DHA) was used to assay βadrenergic receptors from frog plasma membrane (Limbird and Lefkowitz, 1977), turkey erythrocyte ( Vovquelin et al., 1997) and rat erythrocyte (Limbird et al., 1980). Possibly due to the relatively low specific activity of 3HDHA, this radioligand has not been used to study βadrenergic receptors solubilized from other tissues. Due to its slow rate of dissociation from the receptors prior to solublization with a number of different detergents, radiohydroxy derivative of indole (*IHY derivative of indole) was not successfully used in an equilibrium binding assay of βadrenergic receptors following solublization of receptor enriched membranes from a variety of tissues, although (*IHY derivative of indole) has been an excellent probe of adrenoceptor function in certain tissues (Brown and Aurbach, 1976). Yindole labeled with iodine125 has been used extensively a probe for characterizing the binding properties of βadrenergic receptors. The iodinated Y indole retain its high affinity for the receptor in intact rat brain, rabbit left ventricle membrane (Bernadette et al., 1986). Recent spectral and nuclear magnetic resonance studies of iodohydroxyYindole indicated that the iodine atom incorporated into the indole ring at C3 position rather than into the hydroxybenzyl moiety through electrophilic attack of the indole ring. This has been observed in numerous indolecontaining compounds (Houlihn, 1972). According to

59

Coenen et al. iodination of Yindole has been occurred through the replacement of indolenic hydrogen by electrophilic iodination in the activated aromatic system (Coenen et al., 1983) as follow.

III.5.2. Chromatographic identification of 131 IYindole Thin layer chromatography of Y indole Three developing solvents were tested to choose the most useful solvent for separation of iodoYindole from other components in the reaction mixture using TLC method. The three developing solvents were chloroform: acetone: 95% ethanol (20:1:1), Methylene chloride: methanol: concentrated formic acid (150:47:3) and Chloroform: acetic acid: water

(15:4:1). The first solvent separates iodoYindole at R f 0.6 and radioiodide at R f 0.45. This solvent has low resolution (Sungur et al., 1989). The second solvent does not separate iodoYindole from radioiodide and the activity remains at the origin (Spahn et al) . The third solvent separates radioiodide at R f 0.1, iodoYindole at R f 0.7. So the most suitable solvent is the third one. The results obtained were in complete agreement with that previous reported by Zuo and Zhang (1987).

60

HPLC analysis of 131 IY indole Some developing solvents were tested to select the most suitable solvent for the purification of 131 IY indole. Hexane:ethanol:propan2ol mixture (16:3:1) were used as a mobile phase to separate set of beta blockers using RP18 column of HPLC at a flow rate of 1 ml/min. and the retention time of Yindole is 7 min. (Lee et al., 1991). Lacroix et al. (1990) studied the liquid chromatographic determination of Yindole and related compounds in raw materials using acetonitrile: 50mM sodium acetate buffer at pH 5 (7:3) as a mobile phase at a flow rate of 1 ml/min. and their studies resulted in separation of Yindole at retention time of 5.3 min. Sodium bicarbonate: acetonitrile (1:1) separated iodide, Yindole and iodoYindole after retention times of 1.8, 5.8, 10 min. respectively according to Cynthia et al. (1979). The three solvents were evaluated and the later solvent was found to be the most suitable one because of the high resolution between the reaction components. So it is used allover the whole work of chromatographic separation of iodoYindole components. Figures (12), (13) and (14) show the UV absorption chromatogram of KI, Yindole and the reaction mixture, respectively. It was clear that the retention time of KI, Yindole and iodoYindole were approximately, 1.82, 5.8 and 10 min, respectively.

61

Iodide

1.82 min U.V Aborbance,U.V units arbitrary

1 2 3 4 5 6 7 8 910 Retention time,min.

Fig. (12): UV Absorbance of KI after separation on RP18 HPLC column using 0.1 M sod. bicarbonate: acetonitrile (1:1) at a flow rate 1 ml/min.

YIndole

5.8 min U.VAbsorbance, units arbitrary

0 2 4 6 8 10 12 14 Retention time, min.

Fig. (13): UV Absorbance of Yindole after separation on RP18 HPLC column using 0.1 M sod. bicarbonate: acetonitrile (1:1) at a flow rate 1 ml/min.

62

Yindole

5.8 min

iodide IYindole

1.82 min 10 min U.V. Absorbance, U.V. unitsarbitrary

0 2 4 6 8 10 12 14 Retention time, min

Fig. (14): UV Absorbance of mixture of KI and Yindole after separation on RP18 HPLC column using 0.1 M sod. bicarbonate: acetonitrile (1:1) at a flow rate 1 ml/min.

63

III.5.3 Factors affecting the radiochemical yield III.5.3.1 Effect of Yindole concentration The radiochemical yield of 131 IYindole increases with increasing Yindole concentration up to 50 M. Above this concentration there is no significant change. Figure (15) shows that 50M of Yindole is sufficient to achieve 50% radiochemical yield and above this concentration does not affect the radiochemical yield.

III.5.3.2 Effect of pH of the medium In the iodination of Y indole using chloramineT or iodogen, strong pH dependence is found around 6.5 when tagging indole moiety. The influence of pH of the reaction mixture on the percent radiochemical yield of 131 IYindole was studied using phosphate buffer at different pH values 1, 3, 6, 6.5, 7, 7.5, 9 and 10. The results of this study are represented in Figure (16). The data clearly show that a higher radiochemical yield was obtained at pH 7. This is due to the fact that iodide is oxidized easily to iodonium cation I + at pH 7 and indole ring ionized to the anion by the loss of H + ion and the iodination reaction occurs (Coenen et al., 1983). At acidic pH the radiochemical yield of 131 IYindole is very low. At alkaline pH 10, the yield also decreased markedly reaching to 17% and 20.8% when CAT and iodogen were used as oxidizing agents respectively.

64

60

50

40

30

20 Radiochemical yield, % yield, Radiochemical

10

0 0 10 20 30 40 50 60 Yindole concentration, M

Fig. (15): variation of radiochemical yield of [131 I]Y indole with the concentration of Yindole [xM Yindole + 3.7 MBq Na 131 I 10l +25M iodogen], reaction time 30 min, where x is variable.

60

50

40

30

20 CAT Radiochemical yield, % Iodogen 10

0 2 4 6 8 10 pH

Fig. (16): Effect of pH of the reaction medium on the radiochemical yield of [131 I]iodoYindole [50M Y indole + 3.7 MBq Na 131 I 10l +30M oxidizing agent at different pH], reaction time 30 min .

65

III.5.3.3 Effect of oxidizing agent concentration: The radioiodination of Yindole is highly dependent on the concentration of the oxidizing agents. The effect of oxidizing agent concentration on the radiochemical yield of iodoYindole is illustrated in Figure (17). The figure clearly shows that a maximum yield of 39% of iodoYindole was obtained at 30M of CAT. Sharp decrease of the radiochemical yield of iodoYindole was obtained with decreasing the oxidizing agent concentration. The decrease of the radiochemical yield can be mainly attributed to the insufficiency of CAT to oxidize iodide ions to the iodonium ions. Increasing the concentration of CAT above 30M does not increase the yield and may to the formation of oxidative side products (Petzold and Coenon, 1981). In order to obtain a high radiochemical yield of 131 IYindole the quantity of CAT used must be carefully optimized to prevent the formation of other radioactive side products. The iodination of Yindole using iodogen as an oxidizing agent exhibits that the optimum iodogen concentration require to produce the highest radiochemical yield of iodoYindole was 25M. This concentration of iodogen (25M) gives a radiochemical yield of 49% for 131 IYindole. Increasing the iodogen concentration does not affect the radiochemical yield of 131 IYindole.

III.5.3.4. Effect of reaction time: The radiochemical yield was determined at different time intervals using the optimum molar ratio (1:2, 1:1) Yindole: CAT and Yindole: iodogen, respectively and optimum pH value. The results obtained are illustrated in Figure (18). It could be concluded that the percent

66 radiochemical yield increases with increasing the reaction time and completed after 30 min. reaching 39% and 50% for CAT and iodogen respectively. The mild radiochemical yield of 131 IYindole can be explained on the basis of the higher reactivity of the pyrrole ring of indole compared with benzene. This is in complete agreement with the results obtained by Coenen et al. (1983) Increasing the reaction time above 30 min. does not affect the radiochemical yield.

III.5.4 Optimum conditions suggested for the preparation of pure 131 I Yindole: The studies mentioned before reveals that the best reaction conditions for obtaining higher radiochemical yield within 30 min. are 50M of Yindole, 25M of iodogen in phosphate buffer pH 7. After the completion of the reaction 131 IYindole must be separated from the other reaction components and finally the optimized radiochemical yield must be obtained. Figure (19) shows the optimum labeling conditions of Yindole with 131 I using the HPLC analysis of the reaction mixture.

67

60

50

40

30

20

Iodogen Radiochemicalyield,% 10 Cat

0 0 5 10 15 20 25 30 35 40 45 Oxidizing agent concentration, M

Fig. (17): variation of radiochemical yield of [ 131 I]iodoYindole with the concentration of oxidizing agents [50M Y indole + 3.7 MBq Na 131 I 10l +xM oxidizing agent in phosphate buffer pH 7], reaction time 30 min, where x is variable

60

50

40

30

20

Radiochemical% yield, Iodogen CAT 10

0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 Reaction time,min

Fig.(18): Effect of reaction time on the radiochemical yield of [131 I] iodoYindole [50M Y indole + 3.7 MBq Na 131 I 10l +25M oxidizing agent in phosphate buffer, pH 7]

68

III.5.5 Invitrostability of the labeled 131 IYindole: After optimizing the radiochemical yield of 131 IYindole the stability of the labeled product was studied in order to determine the suitable time for injection, in other words, to avoid the formation of undesired products resulted from the radiolysis of the labeled products. The undesired products may be toxic or accumulated in undesired organ. Table (9) shows the stability of 131 IYindole. The stability of the labeled product was followed by TLC using chloroform: acetic acid: water (15:4:1) as developing solvent. From the Table it is obvious that the suitable time for injection is the first three hours after labeling at which no radiolysis takes place. After that the radiolysis of 131 IYindole is very small and this indicates that the labeled product is highly purified from the other reaction components such as oxidizing agents which may catalyze the radiolysis process.

III.5.6 Biodistribution of 131 IYindole in mice Yindole is an adrenoceptor antagonist, and the biodistribution of its labeled compound was found in the organs containing this type of receptors (Yum et al., 2009). The organs investigated include brain, heart and lung of the mouse as clearly shown from Table (10). It is clear that the radioactivity localized in brain; heart and lung are equal to 1.28%, 4.6% and 2.3%, respectively at 45 minutes post injection. The uptake of 131 IYindole through blood brain barrier is lower than heart and lung.

69

X 10 4 7

131 IYindole 6 10 min

5

4

3 Counts / min. / Counts 2 131 I 1.82 min 1

0 0 5 10 15 20 25 30 Retention time, min

Fig.(19): HPLC analysis of a reaction mixture [50M Y indole + 3.7 MBq Na 131 I (10l) +25 M iodogen in phosphate buffer, pH 7] reaction time 30 min.

70

Table (9): Invitrostability of [ 131 I] Yindole.

Time (h) Yield (%) Time (h) Yield (%) 1 53 ± 0.5 9 48.5 ± 1.4 2 53 ± 0.6 18 45.4 ± 1.3 3 52 ± 0.8 20 44.7 ± 0.9 4 51.4 ± 1.2 21 44.3 ± 1.6 5 51.2 ± 1.4 22 43.1 ± 1.2 6 50.8 ± 1.7 23 43 ± 1.7 7 49.3 ± 1.3 24 43 ± 1.9

Table (10): Biodistribution of [ 131 I] –Y indole in mice . Injected Dose/organ organ 15min. 30 min. 45 min.

Blood 25.8 + 0.15 21.2 +0.05 15.3 +0.05 Bone 3.4 + 0.18 2.9 + 0.05 2.6 + 0.06 Muscles 4.8 + 0.66 3.92 +0.27 3.5 + 0.07 Brain 1.1 + 0.18 1.19 +0.17 1.28 +0.13 Kidney 6.2 + 0.09 8.5 +0.09 13.1 +0.02 Heart 6.7 + 0.10 5.2 + 0.11 4.6 + 0.12 Liver 2.1 + 0.05 2.4 + 0.02 2.7 + 0.01 Spleen 1.1 + 0.06 1.5 + 0.04 1.81 + 0.03 Lung 0.9 + 0.1 1.8 +0.03 2.3 + 0.22

71

III.6 Radioiodination of Epidepride III.6.1 introduction In [131 I]Iodoepidepride, the iodine substituted analogue of isoremoxipride, both of which are very potent dopamine D 2 antagonists and its structure as shown.

H N CONHCH 2 C H HO OMe 2 5

131 I OMe

Epidepride was radioiodinated using different oxidizing agents such as chloramineT, iodogen and hydrogen peroxide. CAT is a powerful oxidizing agent compared to both iodogen and hydrogen peroxide so that the side products especially the chlorinated epidepride decreases the radiochemical yield. This chlorinated epidepride is low in case of iodogen and is not observed in case of the non chlorinated oxidizing agent hydrogen peroxide. TLC and HPLC methods were used to analyze reaction component and to estimate both the radiochemical yield and purity. The reaction parameter such as reaction time, pH, epidepride concentration and oxidizing agent concentration were studied to optimize the radiochemical yield and purity. The optimized radiochemical yield was about 90% and the radiochemical purity of the final product was 99.9%. Neuroleptic agents have affinity for dopamine receptors and are therefore potential candidates for radioligand and brain imaging agents. With the discovery of potent substituted benzamides such as eticlopride (Hall et al., 1985). Radioligands

72 with drug receptor dissociation constants K D of < 0.1 nM and become 3 available.[ H] Emonapride [KD2060pM] labels dopamine D 2 receptors in the rat frontal cortex (Kazawa et al.,1990), an area of the brain that has become the focus of interest because of its implication in the action of neuroleptic agents (Lidow et al.,1989). Thus, the possibility of detecting clinically relevant blockade of dopamine receptors in extrastraital area of the brain, where the dopamine D2 receptor density is one to two orders of magnitude less than that in the striatum . Initial studies in man with the iodine123 substituted analogue of raclopride, [ 123 I] IBZM, have demonstrated selective uptake in the basal ganglia compared to cerebellum of 1.5:1 at 1 h post injection (Kung et al., 1990) The discovery of a series of substituted benzamides, structurally related to remoxipride, that are very potent antagonists of apomorphine induced behavior in the rat has presented the potential for developing new and more potent radioligands. One of these compound, the iodine substituted analogue isoremoxipride (epidepride) has the ability to displace [ 3H] spiperone in rat striatal homogenate with an IC50 value of 0.7 nM (Hogberg et al., 1990). Epidepride and its analogues have been discovered a high – affinity radioligands for imaging extrastriatal dopamine D 2 receptors in human brain (Depaulis, 2003) The reaction of Na 131 I with the tributyltin derivatives was made and the radiochemical yield of only 40% was obtained. For brain imaging studies, specific activities ranging from 2,000 to 8,000 Ci/mM were considered sufficiently high for the accurately quantifying the receptor binding without saturation (Clanton et al., 1991).

73

III.6.2 Chromatographic identification of 131 Iepidepride Thin layer chromatography of epidepride After the completion of the reaction, a small spot of the reaction mixture was put on the origin of the TLC sheet, allowed to dry and then developed using ethanol: ethyl acetate (1:1) then cut to similar strips and then counted using γ counter. The separation results indicate that the free iodide 131 has R f of 0.55 and Iepidepride has R f of 0.1. The percentage radiochemical yield was estimated as the ratio of the radioactivity of iodoepidepride to the total activity multiplied by 100. HPLC analysis of epidepride The reaction was neutralized completely, Each component in the reaction mixture was injected onto RP18 HPLC column using an eluent of phosphate buffer (0.1 M, pH 7.4): ethanol (30:70) and a flow rate of 1ml/ min. The reaction mixture was also injected onto RP18 HPLC column for analysis. Figures (20), (21) and (22) show the UV absorption chromatogram of KI, epidepride and the reaction mixture, respectively. It was clear that the retention time of KI, epidepride and iodoepidepride were approximately, 1.2, 8.3 and 12 min, respectively.

74

3000

2500 iodide

2000 1.2 min

1500

1000

500Absobance,U.V arbitrary units

0 0 2 4 6 8 10 12 14 16 Retention time, min

Fig. (20): UV absorbance of KI after separation on RP18 HPLC column using 0.1 M phosphate buffer pH 7.4 : ethanol (30:70) at a flow rate 1 ml/min.

epidepride

8.3 min U.V.Absorbance,units arbitrary

0 2 4 6 8 10 12 14 16 Retention time, min.

Fig. (21): UV absorbance of epidepride after separation on RP18 HPLC Column using 0.1 M phosphate buffer pH 7.4 : ethanol (30:70) at a flow rate 1 ml/min.

75

2500

2000 iodoepidepride iodide 12 min 1500 epidepride 1.2 min 8.3 min 1000

500 U.VAbsorbance, arbitraryunits

0 0 2 4 6 8 10 12 14 16 Retention time, min

Fig. (22): UV absorbance of mixture of KI and epidepride after separation on RP18 HPLC column using 0.1 M phosphate buffer pH 7.5: ethanol (30:70) at a flow rate 1ml/min.

76

III.6.3 Factors affecting the radiochemical yield III.6.3.1 Effect of epidepride concentration

Figure (23) shows that in the presence of H 2O2 as an oxidizing agent the radiochemical yield of [ 131 I]iodo epidepride increases with increasing of epidepride concentration. 10M of epidepride is sufficient to achieve a reasonable labeling yield. Greater amounts of epidepride do not affect the radiochemical yield.

77

100

80

60

40 Radiochemicalyield, % 20

0 0 2 4 6 8 10 12 14 16 concentration of epidepride, M

Fig. (23): Effect of tributyltin epidepride concentration on the 131 Radiochemical yield of [ I] iodoepidepride using H 2O2 as oxidizing agent.

78

III.6.3.2 Effect of pH of the medium The influence of pH of the reaction mixture on the percent The radiochemical yield of the reaction of tributylstannyl epidepride with 131 I using H 2O2 as oxidizing agent using phosphate buffer at different pH values 1,2,3.4,5,6,7 and 9. The results of this study are represented in Figure (24). The data clearly shows that the radiochemical yield increases up to 3.4 and decreases there after gradually to reach the lowest value at pH 9.

III.6.3.3 Effect of reaction time Figure (25) shows the effect of the reaction time on the radiochemical yield of [ 131 I] iodo epidepride. It is found that in case of chloramineT, the reaction is faster than in case of hydrogen peroxide because of the high oxidizing power of chloramineT. But the radiochemical yield is lower in case of chloramineT than H 2O2 this due to formation of radioactive side products especially chlorinated compounds which are clearly observed in figure (26). The optimum reaction times for chloramineT and hydrogen peroxide as oxidizing agents were 10 min. and 20min, respectively.

79

100

80

60

40 Radiochemical% yield, 20

0 0 2 4 6 8 10 p H

Fig. (24): Effect of the pH on the radiochemical yield of 131 [ I]iodoepidepride using H2O2 method.

100

80

60

40 Radiochemicalyield, % 20 CAT

H 2O2

0 0 5 10 15 20 25 30 Reaction Time, min.

Fig. (25): Effect of the reaction time on the radiochemical yield of [ 131 I]Iodoepidepride using chloramineT and iodogen as oxidizing agent.

80

III.6.3.4 Effect of oxidizing agent concentration: It is observed that the use of a 30% hydrogen peroxide solution resulted in an unacceptable low yield of [ 131 I]epidepride (3031%).The use of 3% hydrogen peroxide solution resulted in greater than 95% crude radiochemical yield and 97% radiochemical yield after HPLC purification.

As shown in Table (11), hydrogen peroxide (H2O2) and CAT were compared as oxidizing agents at pH 3.4. The results show that the radiochemical yield in case of H 2O2 is greater than that in case of the chlorinated oxidizing agent (CAT). This can be explained by the high reactivity of the epidepride towards the electrophilic substitution reaction due to the presence of two methoxy group which increase the electron density on the aromatic ring. This high reactivity permits the formation of different undesired side product like chlorinated products especially when using chlorinated oxidizing agents like CAT as shown in Figure (26). From this Figure, the data show that the chlorinated side products are high in case of chloramineT. The main side product was the chloroepidepride which appears at Rt of 11.8 min. This is in complete agreement with the data published by Clanton et al. (1991). To prevent the formation of these side products, the soft H 2O2 was used as oxidizing agent. Figure (22) shows the cold iodination of epidepride using

H2O2. There is a little amount of side products were obtained compared to that in case of chloramineT method.

81

Table (11): Variation of the radiochemical yield of [ 131 I]iodoepidepride with the oxidizing agents concentration.

H2O2(%) Yield (%) CAT Yield (%) at 20 min (nM) at 10 min 1 55.2±0.3 10 30.1±0.2 3 95.7±0.2 20 42.5±0.7 5 88.6±0.7 30 56.3±0.4 10 70.6±0.5 40 68.9±0.1 15 58.3±0.8 50 72.5±0.5 20 42.5±0.3 60 74.2±0.3 30 31. ±0.6 70 69.1±0.2

III.6.4 Optimum conditions suggested for the preparation of pure 131 I epidepride: The studies mentioned before reveal that the best reaction conditions for obtaining higher radiochemical yield within 20 min. are 10 M of epidepride, 10 l of 3% of hydrogen peroxide in a reaction pH value 3.4. After the completion of the reaction 131 Iepidepride must be separated from the other reaction components and finally the optimized radiochemical yield was 99%. Figure (27) shows the optimum labeling conditions of epidepride with 131 I.

III.6.5 Invitrostability of the labeled [131 I]epidepride: After optimizing the radiochemical yield of [ 131 I]epidepride the stability of the final product was studied in order to determine the suitable time for injection, in other wards, to avoid the formation of the undesired products resulted from the radiolysis of the labeled products. The

82 undesired products may be toxic or accumulated in undesired organ. Table (12) shows the stability of 131 Iepidepride. The stability of the labeled product was followed by TLC using ethanol: ethyl acetate (1:1) as developing solvent. From the table it is obvious that the suitable time for injection is the first five hours after labeling at which no radiolysis takes place. After that the radiolysis of 131 Iepidepride is very small and this indicates that the labeled product is highly purified from the other reaction components such as oxidizing agents which may catalyze the radiolysis process.

Table (12): Invitrostability of [ 131 I] iodoepidepride .

H2O2 Time (h) Yield (%) 1 94.5 + 0.2 5 94.4 + 0.4 10 88.9 + 0.5 15 69.1 + 0.7 20 65.5 + 0.3 24 45.8 + 0.6

83

50

40 Iodide

1.2 min epidepride Iepidepride 30 8.3 min 12 min

20 Clepidepride

CAT 11.8 min

10 3 min U.V.absorbance, arbitrary units

0 0 2 4 6 8 10 12 14 16 Retention time, min.

Fig. (26): UV absorbance of the cold reaction of KI and tributyltin epidepride and CAT after separation on RP18 HPLC column using 0.1 M phosphate buffer pH 7.4: ethanol (30:70) at a flow rate 1 ml/min.

X10 4 7

131 Iepidepride 6

12 min 5

4

3

2 131 I Radiochemicalyield, % 1.2 min 1

0 0 2 4 6 8 10 12 14 16 18 20 22 24 Retention time, min

Fig. (27): HPLC analysis of a reaction mixture [10M epidepride + 3.7 MBq 131 Na I 10l +3% H2O2 in phosphate buffer, pH 7.4] reaction time 20 min.

84

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91

ا ا

ه ا إ أاء :

ا اول : : ها ا ء اى اع اص ء اد و اآ ااً وا ا و ا وت ه ا آ ا اوى وا ا ، و ه ا اء ا ااص ا وا ام واد اق ا اد ا أ آ ار م ا ا اف و ادت ا. ها ا أً ق ا ا د ا ال ا و ام اا اآة ارا ت وادو وق أآ ارو . آ أً ق و و ر ادة آت ا د ا م ه اآت وج ا ااض . ا ن إاء ا ر ر ادة س اوة ا واوة اادآ واوة ا ا اى وا وا او واز اى آ وة وأن وآءة ه ات اد ا اوى آ اه . .

ا ا : :

ها ا ت اوت وااد ا واة وأ ا ا ارا آ ها ا اج اد ١٣١ ام ا اف . .

١ و ها ا أ اق د ١٣١ ا و ه ال ا و آ اول و ا ا . .

ه ا ا ا و ق ا اوا وه : :

١ آوا ا ا . .

٢ و اآت ا ام ز ا وا ا اداء.

ا ا : :

و ها اء اد ١٣١ أآ ارم ام ا اف وآ ا آ : :

أ در ارة ن ه ٧٠٠ در ل أ إ ( ١٠٣ آرى ) اد ١٣١ ، أ ز اف ٣٠ د ل ا آ م ٠٨ وام/ ا ا اد ا ات ا . آ آن أ آ روآ ادم ٠١ . .

آ ااء ارات ا ادة ا (Na 131 I ) وآ , 99.9% %98.7 وة ا ا اى،اوة ااد آ ا و اوة ا ام ا ا ٠٨ وام/ . .

و ارا أ اام اد ١٣١ ا ال او آت ا وه آ اول ا م

٢ ام و ا و ا و ا ا ام ا وج ا اش . ه ا آ اد ا ادوم ام اا اآة آ را ت و ادو وق اآ ارو . و ها ا وف ا آ ااد ااد واس ارو وآ ا اآ و ز ا و اوف ا ل ا أ ال ا ٥٠ % دو اول ى ا ل ات ( ٠١ ل ) ذو ا ارو ٧ ة آ و آ اوف ا ل ٩٥% ا ى ا ا ارو ٣٤ ة د آ . و ان در اوة آت ا ٩٩ %. %.

آ درا در ت ها اآ ار ا ا ا . آن ادو اول ل اث ت او، أ آ ادو اا ل ا ت او . .

آ ادو اول ان ارب ن آ اآ ا ا ، ا و ا ١٢٨ ،% ٤٦ % ، ٢٣ % ا . .

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