radiolabeling of some dopamine receptor ligands as models
TRANSCRIPT
Radiolabeling of Some Dopamine Receptor Ligands as Models for BrainImaging
Thesis Presented by
Veronia Refaat Ragheb Ayoub
(Pharmacist)Hot Labs Center (HLC),
Egyptian Atomic Energy Authority (EAEA)
For the degree of masterIn Pharmaceutical Sciences (Pharmaceutics)
Under Supervision of
Prof. Dr. Ahmad Shawky Geneidi
Professor of Pharmaceutics and Industrial Pharmacy
Department of Pharmaceutics and Industrial Pharmacy,
Faculty of Pharmacy,
Ain Shams University.
Prof. Dr. Mohamed Abd El Motaleb Abd El Rahman
Professor of Radiochemistry,
Labeled Compounds Department,
Hot Labs Center,
Egyptian Atomic Energy Authority.
Ass. Prof. Dr. Ismail Taha Ibrahem
Assistant professor of Radiopharmaceutial Chemistry,
Labeled Compounds Department, Hot Labs Center,
Egyptian Atomic Energy Authority.
Department of Pharmaceutics
Faculty of Pharmacy
Ain Shams University
2016
ACKNOWLEDGEMENT
First and foremost thanks to God for helping me to fulfill this
work.
I would like to express my deep gratitude and appreciation to Prof.
Dr. Ahmad Shawky Geneidi, Prof. of Pharmaceutics, Faculty of
pharmacy, Ain Shams University, for kindly supervising this work, for
available help and careful revision of this work. I owe him a great deal
for giving me all the benefits of his experience and academic knowledge;
I will not be able to do his justice
I am profoundly grateful to Prof. Dr. Mohamed Abd El-Motaleb,
Professor of Radiochemistry, Hot Labs Center, Egyptian Atomic Energy
Authority for suggesting the topic, planning of the experimental work,
offering technical facilities and effective supervision through the whole
work.
I am sincerely grateful to Prof. Dr. Ismail Taha, for his effort,
continues advice and support during the course of the thesis.
I am sincerely grateful to my father, mother, husband, son and
the rest of my family for their great help and continuous support and
encouragement.
Finally I would like to thank everyone else at the Hot Labs. Center,
Atomic Energy Authority, whose names are too numerous to mention for
their help in this work
Veronia Refaat
I
Contents
List of Abbreviations ………………………………………………………………………………………………………….
List of Tables …………………………………………………………………………………………………………………………….
List of Figures …………………………………………………………………………………………………………………………..
Abstract ……………………………………………………………..………………………………………………………………………….
Introduction………………………………………………………………………………………………………………………………...
1. Radiopharmaceuticals……………………………………………………………………….………………
1.1 Definition……………………………………………………………………………………………….………………...
1.1.1 Vehicle molecules ………..……………………………………………….…………………
1.1.2 Radionuclides ……………….………………………………………………….…………………
1.2 Radionuclides used for labeling ………………………………………………………………
1.3 Development of new radiopharmaceuticals …………………………...………..
1.3.1 Factors influencing the formulation of
radiopharmaceuticals …………………………………………………………………………………….
1.4 Requirements of good radiopharmaceutical used for
diagnostic purpose ……………………………………………………………………………………………
1.5 Quality control of radiopharmaceuticals ………………………………………….
1.5.1 Physicochemical tests …………………………………………………………………...
1.5.2 Biological tests ………………………………………………………………………………..…
2. Nuclear medicine ……………………………………………………………………………………………….…..
2.1 Therapeutic radiopharmaceuticals ………………………………………………………...….
2.2 Diagnostic radiopharmaceuticals ………………………………………………………............
3 Labeling of pharmaceutical compounds with technetium-99m.
3.1 Methods of labeling with 99mTc …………………………………………………………….……
4 Radioiodination of pharmaceutical compounds ……..……………………………
4.1 The main developed instrumentation for iodine production …..
5 Brain imaging…………………………………………………………………………………………………………………….
5.1 Brain imaging techniques …………………………………………………………………………...….
5.2 Gamma camera …………………………………………………………………………………………………...…
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5.3 The blood brain barrier ………………………………………………………………………………….…
5.3.1 Fundamentals of BBB …………………………………………………………………..
5.3.2 BBB permeation mechanisms …………………………………………………
6 Dopamine Receptors …………………………………………………………………………………………………..
6.1 Dopamine receptor subtypes, localization and function ……………
6.1.1 D1-Like Receptors ………………………………………………………………………….
6.1.2 D2-Like Receptors ………………………………………………………………………….
6.2 Radiopharmaceutical drugs used in imaging the
dopaminergic system …………………………………………………………………………………………
Scope of Work ………………………………………………………………………………………………………………………….
Chapter one ………………………………………………………………………………………………………………………………..
Introduction ……………………………………………………………………………………………………………………………….
Experimental …………………………………………………………………………………………………………………………….
1 Materials ………………………………………………………………………………………………………………………………
1.1 Chemicals …………………………………………………………………………………………………………………
1.2 Equipment ……………………………………………………………………………………………………………….
1.3 Radioactive material …………………………………………………………………………………………
1.4 Animals ………………………………………………………………………………………………………………………
Methodology ……………………………………………………………………………………………………………………………...
1 Preparation of stock solution of SnCl2.2H2O ………………………………………….
2 Kit preparation and labeling of different ligands with
technetium-99m ………………………………………………………………………………………………………….
2.1 99mTc-ropinirole ……………………………………………………………………………………………………
2.2 99mTc-itopride …………………………………………………………………………………………………………
3 Analysis of the reaction mixture (radiochemical yield) …………………
3.1 Ascending paper chromatographic analysis ……………………………………..
3.2 HPLC analysis of the prepared 99mTc-ligands complexes ….…...
3.2.1 99mTc-ropinirole complex …………………………………………………………...
3.2.2 99mTc-itopride complex ………………………………………………………………..
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4 Study of the factors affecting the percent radiochemical yield
of 99mTc-ligand complexes ………………………………………………………………………………………
4.1 Effect of pH of reaction medium ………………………………………………………………
4.2 Effect of reducing agent amount ………………………………………………………………
4.3 Effect of ligand amount …………………………………………………………………………………
4.4 Effect of reaction time ……………………………………………………………………………………...
4.5 Effect of reaction temperature ……………………………………………………………………
4.6 In vitro stability study ……………………………………………………………………………………….
5 Determination of the partition coefficient for 99mTc-ligand
complexes ……………………………………………………………………………………………………………………………..
6 Statistical analysis ……………………………………………………………………………………………………….
7 In-vivo biodistribution of 99mTc-ligands ………………………………………………………
8 Blocking study of dopamine receptor …………………………………………………………
RESULTS AND DISCUSSION …………………………………………………………………………………
1 HPLC analysis ……………………………………………………………………………………………………………
2 Study of the factors affecting the percent radiochemical yield
of 99mTc-ligands complexes …………………………………………………………………………………….
2.1 Effect of pH of reaction medium ………………………………………………………………
2.2 Effect of reducing agent amount ………………………………………………………………
2.3 Effect of ligand amount …………………………………………………………………………………...
2.4 Effect of reaction time ……………………………………………………………………………………...
2.5 Effect of reaction temperature …………………………………………………………………….
2.6 In -vitro stability study …………………………………………………………………………………….
2.7 Determination of the partition coefficient for 99mTc-ligand
complexes …………………………………………………………………………………………………………………
2.8 In-vivo biodistribution of 99mTc-ligand …………………………………………………
2.9 Blocking study of dopamine receptor ……………………………………………………
Conclusions………………………………………………………………………………………………………………………………….
Chapter Two ……………………………………………………………………………………………………………………………..
Introduction ……………………………………………………………………………………………………………………………….
Experimental …………………………………………………………………………………………………………………………….
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1 Materials ………………………………………………………………………………………………………………………………
1.1 Chemicals …………………………………………………………………………………………………………………
1.2 Equipment ……………………………………………………………………………………………………………….
1.3 Radioactive material …………………………………………………………………………………………
1.4 Animals ………………………………………………………………………………………………………………………
Methodology ……………………………………………………………………………………………………………………………...
1 Radioiodnation method of prochlorperazine ………………………………………….
2 Analysis of the reaction mixture (radiochemical purity and
yield) …………………………………..…………………………………………………………………………………………………….
2.1 Ascending paper chromatographic analysis ………………………………………
2.2 HPLC analysis of radiochemical purity of the prepared 125I-
prochlorperazine …………………………………………………………………………………………………
3 Study of the factors affecting the percent radiochemical yield
of 125I-prochlorprazin ………………………………………………………………………………...………………
3.1 Effect of pH of reaction medium ………………………………………………………………
3.2 Effect of oxidizing agent (Chloramine-T) amount ………………………
3.3 Effect of substrate amount ………………………………………………………..……………………
3.4 Effect of reaction time ……………………………………………………………………………………...
3.5 Effect of reaction temperature ……………………………………………………………………
3.6 In-vitro stability …………….…………………………………………………………………………………….
4 Determination of the partition coefficient for 125I-
prochlorperazine……………………………………………………………………………………………………………..
5 Statistical analysis ……………………………………………………………………………………………………….
6 In-vivo biodistribution of 125I-prochlorperazine ………………………………...…
7 Blocking study of dopamine receptor …………………………………………………………
RESULTS AND DISCUSSION …………………………………………………………………………………
1 HPLC analysis of radiochemical purity ………………………………………………….
2 Study of the factors affecting the percent radiochemical yield
of 125I-prochlorperazine ………………………………………………………………………………………….
2.1 Effect of pH of reaction medium ………………………………………………………………
2.2 Effect of oxidizing agent (Chloramine-T) amount ………..………………
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2.3 Effect of substrate amount ……………………………………………………………………………...
2.4 Effect of reaction time ……………………………………………………………………………………...
2.5 Effect of reaction temperature …………………………………………………………………….
2.6 In -vitro stability study …………………………………………………………………………………….
2.7 Determination of the partition coefficient for 125I-
prochlorperazine ……………………………………………………………………………………………...……
2.8 In-vivo biodistribution of 125I-prochlorperazine ………………………………
2.9 Blocking study of dopamine receptor ……………………………………………………
Conclusions………………………………………………………………………………………………………………………………….
Summary ……………………………………………………………………………………………………………………………….……..
References …………………………………………………………………………………………………………………………….……..
Appendix …….……………………….…………………………………………………………..…………………………………….……..
Arabic Summary ………………………………………………………………………………………………………………...…
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و- أ
VI
List of Abbreviations
ANOVA Analysis of variance
C Carbon element
CAT Chloramine-T (N-chloro-p-toluene sulfonamide sodium salt)
CPM Count per minute
CT Computed tomography
ECD Ethyl cysteinate dimer
ET Emission tomography
HMPAO Hexamethyl propylene amine oxime
I Iodine
I.D. Injected dose
I.V. Intravenously
IMP Isopropyl iodoamphetamine
KeV Kilo electron volt
MAG3 Mercapto-acetyl glycyl glycyl glycine
M.Wt Molecular weight
MBq Mega Becquerel
Mo Molybdenum element
MRI Magnetic resonance imaging
N2 Nitrogen gas
Na Sodium element
Na99mTcO4 Sodium pertechnetate
NaBH4 Sodium borohydride
NaCl Sodium chloride salt
P Level of significance
p.i. Post injection
PC Paper chromatography
VII
PET Positron emission tomography
pH Power of hydrogen
Rf Retension factor
RH-99mTc Reduced-hydrolyzed Technetium-99m (colloid)
S.D Standard deviation
Sn Stannous element
SPECT Single photon emission computed tomography
T/NT Target/Non-Target
t1/2 Half life
Tc Technetium
te Effective half-life
tP Physical half-life
UV Ultra violet
α Alpha particles
β Beta particles
γ Gamma emission
VIII
PageTable titleTableno.
12Examples of some radiopharmaceuticals used astherapeutic agents
I
14Examples of radiopharmaceuticals used as diagnosticagents
II
16Essential groups present in many chelating agentsused for 99mTc complexation
III
21Production methods and radioactive properties of123I, 125I and 131I
IV
27Differences between PET and SPECTV57Radiochromatographic behavior of different
radiochemical species and their Rf values for 99mTc-ligands complexes
1
63Retention times values in HPLC chromatograms of99mTc-ropinirole and 99mTc-itopride
2
66Effect of pH value of the reaction mixture on thepercent radiochemical yield of 99mTc-ropinirolecomplex
3
67Analysis of variance of the percent radiochemicalyield of 99mTc-ropinirole at different pH values
4
68Effect of pH value of the reaction mixture on thepercent radiochemical yield of 99mTc-itopride complex(Using SnCl2)
5
69Analysis of variance of the percent radiochemicalyield of 99mTc-itopride at different pH (Using SnCl2)
6
69Effect of pH value of the reaction mixture on thepercent radiochemical yield of 99mTc-itopride complex(Using NaBH4)
7
70Analysis of variance of the percent radiochemicalyield of 99mTc-itopride at different pH (Using NaBH4)
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72Effect of Sn(II) amount on the percent radiochemicalyield of 99mTc-ropinirole complex
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73Analysis of variance of the percent radiochemicalradiochemical labeling yield of 99mTc-ropinirole atdifferent amounts of Sn(II)
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74Effect of Sn(II) amount in the reaction mixture on thepercent radiochemical yield of 99mTc-itopride complex
11
75Analysis of variance of the percent radiochemicallabeling yield of 99mTc-itopride at different amountsof Sn(II)
12
List of tables
IX
75Effect of NaBH4 amount in the reaction mixture onthe percent radiochemical yield of 99mTc-itopridecomplex
13
76Analysis of variance of the percent radiochemicallabeling yield of 99mTc-itopride at different amountsof NaBH4
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77Effect of ropinirole amount on the percentradiochemical yield of 99mTc-ropinirole complex
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78Analysis of variance of the percent radiochemicallabeling yield of 99mTc-ropinirole at different amountsof ropinirole
16
79Effect of itopride amount in the reaction mixture onthe percent radiochemical yield of 99mTc-itopridecomplex (Using SnCl2)
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80Analysis of variance of the percent radiochemicallabeling yield of 99mTc-itopride at different amountsof itopride (Using SnCl2)
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80Effect of itopride amount in the reaction mixture onthe percent radiochemical yield of 99mTc-itopridecomplex (Using NaBH4)
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81Analysis of variance of the percent radiochemicallabeling yield of 99mTc-itopride at different amountsof itopride (Using NaBH4)
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82Effect of reaction time on the percent radiochemicalyield of 99mTc-ropinirole complex
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83Analysis of variance of the percent radiochemicallabeling yield of 99mTc-ropinirole at different reactiontime.
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84Effect of reaction time on the percent radiochemicalyield of 99mTc-itopride complex (Using SnCl2)
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84Analysis of variance of the percent radiochemicallabeling yield of 99mTc-itopride at different reactiontime (Using SnCl2)
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85Effect of reaction time on the percent radiochemicalyield of 99mTc-itopride complex (Using NaBH4)
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86Analysis of variance of the percent radiochemicallabeling yield of 99mTc-itopride at different reactiontime (Using NaBH4)
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87Effect of reaction temperature on the percentradiochemical yield of 99mTc-itopride complex (UsingNaBH4)
27