신경질환에서 산화적 손상의 바이오마커 탐색과 중재요법 연구
Biomarker Identification and Therapeutic Prevention of Oxidative Stress in Neurological Diseases
- 주제(키워드) Stroke , Biomarker , Proteomics , CSF , Oxidative stress , albumin
- 발행기관 아주대학교
- 지도교수 곽병주
- 발행년도 2009
- 학위수여년월 2009. 2
- 학위명 박사
- 학과 및 전공 일반대학원 신경과학기술과정
- 실제URI http://www.dcollection.net/handler/ajou/000000009507
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Oxidative stress plays a major role in tissue damage under pathological conditions and has been targeted for pharmacological prevention of nerve cell injury in a number of neurological diseases including stroke, Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS). Unfortunately, although necessary for the development of new drugs, useful diagnostic biomarkers of oxidative stress for neurological diseases are not yet available. In the first part of the current study, we demonstrated that protein alterations in cerebral spinal fluid (CSF) of stroke patients, including expression and post-translational modifications such as oxidation, were investigated using two-dimensional electrophoresis (2-DE). Protein oxidation was detected with immunoblotting using 2, 4-dinitrophenylhydrazine (2, 4-DNPH) and anti-DNP antibody. In the CSF proteins of stroke patients, approximately 48 spots were altered after the onset of stroke, including alpha-1-acid glycoprotein, alpha-1-microglobulin, vitamin D-binding protein precursor (DBP), beta-actin, transthyretin, and complement C4. In addition, human serum albumin (HSA) was a major oxidized protein found after stroke in CSF. Albumin is well known as an endogenous anti-oxidant in the blood. We suggest that albumin provided protection against reactive oxygen species, preventing neuronal cell death in the CA1 region following 10 min of transient forebrain ischemia. We also found that the function of albumin as an ROS scavenger could be changed by modification of its oxidation status in vivo. A second issue that also needs consideration regarding development of new CNS drugs concerns the ability of various compounds to cross the blood-brain barrier (BBB). In several clinical trials, antioxidant drugs such as vitamin E have shown no beneficial effect because an effective therapeutic concentration could not be achieved in the brain. The BBB penetration rate is intimately associated with success of CNS targeted drug development. Pharmacokinetics and pharmacodynamics studies on the target organ are very important in the process of drug development. In second part of this study, we studied the pharmacokinetics and pharmacodynamics of a new drug candidate, AAD-2004, which has been proposed as having antioxidant properties capable of targeting neurodegenerative diseases. LC-MS was first used to validate pharmacokinetics and pharmacodynamics analysis of AAD-2004. This compound reduced oxidative stress products such as nitrotyrosine and 8-OHdG in ALS mice. Moreover, it showed maximal effects at Cmax = ~ 2.12 μg/ml and AUC = ~3.92 μg.hr/ml in blood that could realistically be expected to achieve more than effective dose in the brain.
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TABLE OF CONTENTS
논문인준서 I
ABSTRACT II
TABLE OF CONTENTS IV
LIST OF FIGULES VIII
LIST OF TABLES X
ABBREVIATION XI
I. INTRODUCTION 1
A. Overview 1
B. Oxidative stress in neurological diseases 1
1. Ischemic stroke 1
2. Alzheimer’s disease (AD) 2
3. Amyotrophic lateral sclerosis 4
C. Monitoring of CSF biomarkers in neurological diseases 5
D. Clinical trials of antioxidants in neurological diseases 5
E. Purpose of study 7
II. MATERIALS AND METHODS 8
A. Materials 8
1. Human CSF 8
2. Animals 8
3. Drugs 8
4. Antibodies 8
B. Methods 10
1. Proteomics 10
(A) Sample preparation 10
(1) For CSF 10
(2) 2-DE 10
(3) Silver Staining 10
(4) In-gel digestion 11
(5) Database searching for protein identification 11
2. Biochemical methods 11
(A) Oxyblot and Western blot analysis 11
(B) Determination of protein carbonyls 12
(C) Albumin Purification 12
(D) Preparation of oxidized plasma proteins 12
3. In vivo methods 12
(A) Transient forebrain ischemia 12
(B) Focal cerebral ischemia 13
(C) Measurement of brain infarction 14
(D) Monitoring of physiological parameters 14
4. HPLC-MS analysis 14
(A) Plasma sample preparation for standard 14
(B) Test System 15
(C) Plasma sample preparation 15
(D) Pharmacokinetic Analysis 15
III. RESULTS 17
A. Proteomic analysis of CSF samples from control and stroke patients 17
B. Identification of oxidized proteins in the CSF from stroke patients 18
C. Anti-oxidant and neuroprotective action of albumin 19
D. Anti-oxidant and neuroprotective action of AAD-2004 in animal models of neurological diseases 21
1. Blockade of oxidative stress by AAD-2004 21
E. Pharmacokinetics/Pharmacodynamics study of AAD-2004 22
1. Validation of analysis of AAD-2004 for HPLC-MS 22
(A) Linearity 22
(B) Precision and Accuracy 22
(C) Sensitivity 22
2. Analysis of plasma concentration of AAD-2004 22
(A) Calibration data 22
(B) Plasma-brain transfer ratio of [14C] AAD-2004 in rats 23
(C) Plasma concentration of AAD-2004 following intraperitoneal (i.p.) injections in C57BL/6 mice 23
(D) Plasma concentration of AAD-2004 following oral administration in C57BL/6 mice 23
(E) Plasma concentration of AAD-2004 following oral administration in G93A mice 24
IV. DISCUSSION 54
A. Searching for biomarkers of stroke in cerebrospinal fluids 54
B. Therapeutic prevention of oxidative stress 58
V. CONCLUSION 60
A. Identification of stroke markers 60
B. Therapeutic prevention of oxidative stress 60
REFERENCES 61
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LIST OF FIGULES
Fig. 1. Diffusion-weighted MRI image of patients with variable infarct patterns. 25
Fig. 2. Validation of plasma contamination of CSF. CSF was used to determine the level of plasma protein contamination.. 27
Fig. 3. Proteomic analysis of protein alternations in the cerebrospinal fluid (CSF) from control and stroke patient group. 28
Fig. 4. MALDI-TOF analysis following in-gel trypsin digestion. 29
Fig. 5. Classification of the altered proteins in CSF from stroke patients 31
Fig. 6. 2D-oxyblot analysis of protein oxidation in CSF of stroke patients.. 33
Fig. 7. Western blot analysis of albumin in CSF of stroke patients 34
Fig. 8. Carbonyl level of albumin in plasma from patients. 35
Fig. 9. Systemic administration of human serum albumin prevents free radical production and delayed neuronal death in the CA1 area after transient forebrain ischemia. 36
Fig. 10. Comparison neuroprotect effect of oxi-albumin and native albumin after systemic administration of human serum albumin following transient forebrain ischemia.. 39
Fig. 11. Prevention of rat plasma protein oxidation by AAD-2004 ex vivo. 40
Fig. 12. Intraperitoneal administration of AAD-2004 blocks oxidative stress in ALS mice. 41
Fig. 14. Calibration curve for AAD-2004 in solution.. 45
Fig. 15. Sample HPLC chromatogram of AAD-2004 and AAD-134. 45
Fig. 16. Calibration curve of AAD-2004 in mouse plasma. 47
Fig. 17. Plasma concentration of AAD-2004 versus time profile after i.p. injection of single 2.5 mg/kg in the C57BL/6 mice. 48
Fig. 18. Plasma concentration of AAD-2004 versus time profile after p.o. of single 2.5 and 5 mg/kg in the C57BL/6 mice. 50
Fig. 19. Plasma concentration of AAD-2004 versus time profile after p.o. of single 5 and 10 mg/kg in the G93A mice. 52
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LIST OF TABLES
Table 1. Diagnosis of Stroke patients 26
Table 2. Increased proteins profile in CSF from stroke patients. 30
Table 3. Diagnosis of Stroke patients 32
Table 4. Physiological measurement parameters 38
Table 5. Calibration curve parameters for AAD-2004 in solution 44
Table 6. Calibration curve data for AAD-2004 in solution 44
Table 7. Calibration Results 46
Table 8. Plasma-brain transfer ratio of [14C]AAD-2004 concentration in rats 47
