Bioassays in Experimental and Preclinical Pharmacology

ISBN-13: 9781071612354 / Angielski / Miękka / 2022

Karuppusamy Arunachalam;Sreeja Puthanpura Sasidharan

Opis

This detailed book explores protocols for a wide array of preclinical pharmacology and toxicology evaluations to be applied to chemical drugs and their development through in vitro, involving tissues and cell lines, and in vivo models, using animals as experimental systems, utilized to conduct pharmacological research. Written for the Springer Protocols Handbooks series, the methodologies included in this collection have been standardized by the authors through extensive use in the lab so that they are ready to be applied in the labs of readers around the world. 

Authoritative and practical, Bioassays in Experimental and Preclinical Pharmacology aims to assist undergraduate and postgraduate students, research scholars, scientists, and other academicians performing research in the vital field of drug discovery.

Spis treści

1.      Overview

2.      Cell Culture Assays

2.1  Cell Cycle Assay: Propidium Iodide Marking

2.2  Apoptosis Assay: Annexin V/Propidium Iodide Marking

2.3  Cytotoxicity in Caco-2 cells by the Alamar blue method

2.4  Viability in Caco-2 cells stimulated with lipopolysaccharide (LPS)

2.5  Determination of Nitric oxide (NO) by in vitro experiment

2.6  Analysis of specific cytotoxicity against RAW 264.7 for determination anti-inflammatory activity in vitro

2.7  Scratching of Fibroblast cells (scratch assay)

2.8  Evaluation of the in vitro inflammatory response in AGS cells

3.      Phytochemical Analysis

3.1  Preliminary phytochemical assays

3.2  Quantitative analysis of secondary metabolites

3.3  Thin Layer Chromatography Analysis

3.4  Analysis by High Performance Liquid Chromatography (HPLC)

3.5  Mass spectrometry

3.6  Electrospray Ionization Mass Spectrometry (ESI-MS) analysis

3.7  Identification of phenolic compounds by HPLC-LC-ESI- MS/MS

3.8  Quantitative determination of total alkaloids content by HPLC

3.9  Quantitative determination of total alkaloids content by HPLC

4.      Preclinical Drug Dose Calculation

4.1  Conversion based on normalization of dose-to body surface area

4.2  Determination of animal equivalent dose

5.      Toxicology Studies (In Vitro and In Vivo)

5.1  Cytotoxicity evaluation using Alamar blue

5.2  Micronucleus test

5.3  Comet test

5.4  Effect on cell cycle

5.5  In vivo acute toxicity study

5.6  Subchronic toxicity (repeated doses)

6.      Standard Procedure for Anesthesia in Preclinical Experiments

6.1  Planning and anesthetic care

6.2  Pre-Procedure

6.3  Experimental Procedure

7.      General Considerations and Collection of Animal Blood

7.1  Vein blood collection

7.2  Tail vein blood collection

7.3  Cardiac puncture blood collection

7.4  Posterior vena cava

7.5  Orbital sinus

8.      Animal Experiments on the Cardiovascular System

8.1  Obtaining electrocardiogram (ECG) and blood pressure (BP) signals

8.2  Myocardial ischemia induction

9.      Animal Experiments on Ulcerative Colitis

9.1  Ulcerative colitis induced by 2,4,6-Trinitrobenzenesulfonic acid (TNBS)

9.2  Chronic ulcerative colitis induced by Dextran Sodium Sulfate (SSD) 2%

9.3  Colitis with recurrence (Chronic)

10.  Experiments on Anti-Bacterial Activities

10.1       Antibacterial activity by in vitro assays

10.2       Animal Experiments on antibacterial activity

11.  Experiments on Anti-Fungal Activities

11.1       Agar Disc Diffusion Method

11.2       Minimum Inhibitory Concentration (MIC)

11.3       Sorbital test

11.4       Neurospora crassa assay

11.5       Effect of Ergosterol on MIC

11.6       Test of Β1,3-D-Glucan Synthase (GS)

11.7       Virulence Factors Inhibition of Candida albicans

11.8       Germinative Tube Inhibition Test

11.9       Biofilm assay

11.10   Time-kill studies

11.11   Determination of Antifungal Activity by in vivo

12.  Animal Experiment on Wound Healing Activity

12.1       Excision wound healing model

12.2       Incision wound healing model

12.3       Evaluation of the effect of the extract on migration/proliferation in fibroblasts

12.4       Surgical wound infected in rats (in vivo)

13.  Animal Experiments of Anti-inflammatory activities

13.1       Inflammatory Paw edema

13.2       Lipopolysaccharide (LPS)-induced peritonitis

13.3       Murine Model of Immediate Hypersensitivity Induced By OVA

13.4       Air pouch inflammation model

13.5       Cotton pellet inflammation model

14.  Animal Experiment on Anti-Nociceptive Activities

14.1       Abdominal constriction by acetic acid

14.2       Formalin test 2.5%

14.3       Hot plate Test

15.  Experiments on Anti-Oxidant Activities

15.1       DPPH free radical scavenging activity

15.2       Hydrogen peroxide radical scavenging activity

15.3       Free radical nitric oxide scavenging activity

15.4       Ferric Reducing Antioxidant Power assay

15.5       Determination of superoxide dismutase (SOD)

15.6       Glutathione peroxidase (GPx) activity

15.7       Determination of catalase (CAT)

15.8       Determination of reduced glutathione (GSH)

15.9       Determination of malonaldehyde (MDA)

15.10   Total protein dosage

15.11   Test of oxidative degradation of deoxyribose

16.  Animal Experiments on Hepatoprotective Activities

16.1       Acute liver damage by paracetamol

16.2       Treatment of acute ethanol-induced injury

16.3       The induction of liver damage by carbon tetra chloride (CCl₄)

17.  Animal Experiments on Anti-Diarrheal Activities

17.1       Gastric emptying

17.2       Intestinal transit

17.3       Enter pooling induced by castor oil

17.4       Microvascular permeability

17.5       Activity on the H⁺/K⁺ pump – ATPase

1. Animal Experiments on Central Nervous System (CNS)

18.1       Stress Induced experiment

18.2       Elevated Cross Maze (ECM)

18.3       Open Field Test (OFT)

18.4       Object Recognition Test (ORT)

18.5       Murinometric evaluation

1. Experiments on Anti-Cancer Activities (In Vitro and In Vivo)

19.1       Cytotoxicity test

19.2       Cell cycle assay

19.3       Assessment of cell viability and proliferation using the MTT method

19.4       Evaluation of clonogenic survival of different cell lines

19.5       In vivo anti-tumor activity

1. Animal Experiments on Anti-Diabetic Activities

20.1       Inhibition of α-amylase

20.2       Inhibition of α-glucosidase

20.3       In vivo Antidiabetic activity

Animal Experiments on Gastric Ulcer

21.1       Gastric Injury Induced by Ethanol

21.2       Gastric Injury Induced by Acidified ethanol (Ethanol/HCl)

21.3       Indomethacin induced gastric injury

21.4       Gastric Injury Induced by Acetic Acid (chronic ulcer) in mice

21.5       NSAID ulcer experiment induced by Piroxicam

21.6       Pylorus ligation experiment in mice

21.7       Stress-induced gastric injury (water restraint)

21.8       Role of NO in a gastric ulcer model pretreated with L-NAME

21.9       Gastric Injury Induced by Ethanol/HCl for quantification of mucus

21.10   Ethanol/HCl-Induced Gastric Lesion with Indomethacin, L-NAME, Glibenclamide, Yohimbine

21.11   Ischemia-reperfusion-induced gastric ulcer

21.12   Evaluation of anti-H. pylori activity in vivo

21.13   Evaluation of anti-Helicobacter pylori activity in vitro

1. Protein Extraction and Western Blot Analysis

22.1       Gel preparation

22.2       Samples application

22.3       Running

22.4       Preparing Membranes

22.5       Revelation

1. Gel Electrophoresis and PCR Amplification

23.1       DNA Extraction

23.2       Dosage of RNA and DNA

23.3       Solutions for Electrophoresis and Western blotting

1. Molecular Docking Methods for Drug Design

24.1       Biological activity calculation

24.2       Molecular docking between the inhibitors and target proteins