1. General Introduction

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1. General Introduction

1.1 Overview of Endocannabinoid System………...5

1.2 How ECs works………...6

1.3 Endocannabinoids………...7

1.4 Endocannabinoid receptors and their synthetic ligands……...9

1.5 MAGL, ABHDs, FAAH, COX-2 and EMT………...15

1.5.1 Mono Acyl Glycerol Lipase (MAGL)………...16

1.5.2 α/β Hydrolases (ABHD-6 and ABHD-12)………...18

1.5.3 Fatty Acid Amide Hydrolase (FAAH)………...19

1.5.4 Cyclooxygenase type II (COX-2)………...20

1.5.5 Endocannabinoid Membrane Transporter………...22

2. Aim of Thesis

3. Materials and Methods

3.1 Materials………...27

3.1.1 Materials used……….…………...27

3.1.2 Cell lines………...28

3.2 Methods………...29

3.2.1 Defrost and maintenance of cell culture………...29

3.2.2 CB1r/CB2r membrane preparation………...30

3.2.3 Quantification of membranes with method of Biureto…….31

3.2.4 [³H]CP-55,940 competition binding Assay………...32

3.2.5 [³⁵S]GTPγS binding assay……….33

3.2.6 MAGL activity inhibition assay………....34

3.2.7 ABHDs activity inhibition assay………...35

3.2.8 FAAH activity inhibition assay………...35

3.2.9 COX-2 inhibition assay………...36

3.2.10 AEA uptake inhibition assay………...36

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3.2.11 2-AG uptake inhibition assay………37

4. Results and Discussions

4.1 Cannabinoid receptor binding………...41

4.1.1 Concentration-dependent binding curves of biphenylic derivatives………...43

4.1.2 Concentration-dependent binding curves of pyridine derivatives………....44

4.2 [³⁵S]GTPγS assay performed for biphenylic derivatives (C) and pyridine derivatives (A and B) on CB1r and CB2r…...48

4.2.1 Functional activity evaluation of biphenylic derivatives....49

4.2.2 Functional activity of pyridine derivatives measured by for [³⁵S]GTPγS………...50

4.3 Inhibition activity on MAGL, FAAH, ABHDs and hCOX-2 activity………...58

4.3.1 Inhibition activity on MAGL………...58

4.3.2 Inhibition activity on ABHDs………..60

4.3.3 Inhibition activity on FAAH………...62

4.3.4 Inhibition activity on hCOX-2………..63

4.4 Uptake inhibition……….67

5. References

~ 3 ~ List of abbreviations

ECs = endocannabinoid system

Δ9-THC = Δ9-tetrahydrocannabinol CBD = cannabadiol

2-AG = 2-arachidonoylglycerol

2-OG = 2-oleoylglycerol

AA = arachidonic acid

ABHD-6, ABHD-12 = α-β-hydrolase domain 6,12

AEA = N-arachidonoylethanolamide

2-AG = 2-arachidonoylglicerol

CB1r, CB2r = cannabinoid receptor 1, 2

hCOX-2 = human cyclooxygenase-2 MAGL = mono acyl glycerol lipase

FAAH = fatty acid amide hydrolase

2-AGE = 2-arachidonyl-glycerylether

Virodhamine = O-arachidonoyl-ethanolamine

NADA = N-arachidonoyl-dopamine

PEA = palmitoyl ethanolamide

OA = oleamide

GABA = γ-Aminobutyric acid

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DSI = depolarization-induced suppression of inhibition

DSE = depolarization-induced suppression of excitation

LTP = long-term potentiation

LTD = long-term depression

CNS = central nervous system

PNS = periphery nervous system

PG = prostaglandin

BSA = bovine serum albumin

GDP = guanosine diphosphate

GTP = guanosine triphosphate

Hot = compound radiolabeled Cold = compound not radiolabeled SR1 = SR141716A

SR2 = SR144528

DMSO = dimethyl sulfoxide

EtOH = ethanol