Laboratory Manual Pharmacology I&II (PHL 313 & 322) Dr/ Mohamed N Ansari Dr/ Majid A Ganaie Department of Pharmacology College of Pharmacy Salman bin Abdulaziz University 1435-1436 / 2014 – 2015 Lab No. TABLE OF CONTENTS PAGE Pharmacology I Laboratory Orientation 2-5 1 Study of Different Laboratory Animals and Their Application 6-7 2 Study of Different Stages of Anesthesia 8-9 3 Study of Some Basic Instruments Used for Isolated Tissue 10-11 Experiments 4 Routes of Drug Administration to Laboratory Animals 12 5 To Evaluate the Analgesic Potency of Drug by Formalin Test 13-15 6 To Evaluate the Analgesic Potency of Drug by Tail Flick Method 16-17 7 To Evaluate the Analgesic Potency of Drug by Hot Plate Method 18-19 8 Screening of Different Diuretics Using Laboratory Animals 20-21 9 Screening of Anti-inflammatory Drug Using Hind Paw Method 22-23 Pharmacology II 24 1 LABORATORY ORIENTATION Introduction: The laboratory portion of this course is designed to study the different laboratory animals, their applications and screening of different pharmacological agents thoroughly than it is presented in lecture. Core learning will come from practical and tissue studies. This method of ‘hands on’ learning should also enhance and strengthen the knowledge you gain in lectures. At times you will be working individually, in pairs or in groups of three or four. Each lab period is loosely structured to begin with a short introduction to the exercise that highlights the activities of the day, what materials are available for use and any changes in procedures. After that you will work independently to learn the material. There is never enough time in lab to go over each and every item that you are assigned. The lab is a designated a time when you have access to materials that you will not have available during home study time. Some of the information assigned in lab you can learn at home, particularly animals, instruments, method, procedure, application, mechanism of action of drugs etc. General Lab Rules: 1. Read the lab exercise before you come to lab. There is not time to review every aspect of each exercise and still give you time to work on your own. I will assume that you know what the exercise covers in general and I will only review changes or specific materials that you will use. 2. Before each lab, use the terminology list to mark the items in your manual’s text and illustrations that you are responsible for learning. 2. Read and memorize the laboratory safety rules of the lab below. The preservatives are irritants and some of you may be allergic to them. Gloves must be used during dissections and will be provided. Your dissecting tools will be provided for you as well. 2 Pharmacology Laboratory: Safety, Procedures, Emergencies These are minimum safety requirements. Instructors may institute additional policies at their discretion. 1. No open food or drink is permitted at any time, whether a lab is in progress or not. No eating, drinking, candy, cough drops, chewing gum or tobacco is permitted. All beverage and food containers must be put away in a backpack/bag/purse or left outside of the lab. There are shelves outside in the hallway to store food and beverages during lab. Never taste anything at all while in the lab rooms, unless it is a part of the lab activity (such as PTC paper). Also, do not apply cosmetics in lab (this includes lip balm). 2. Visitors under 16 years of age and children are not allowed in the lab rooms at any time. Visitors over 16 may be allowed at the instructor’s discretion, as long as the lab activity does not involve hazardous materials. 3. Know the locations of the eye wash and shower stations, fire alarm, fire extinguisher, first aid kit, and emergency exits. Do not block access to these with trash cans, recycle bins, etc. 4. Safety instructions are given at the beginning of each lab period. Always arrive on time so that you know what you are supposed to do and are informed of any specific safety concerns or safety equipment associated with the day’s lab activity. 5. Wear any required personal protective equipment (lab coat, apron, goggles, etc). 6. Stash book bags safely so that they won’t trip people. 7. Report all illnesses, injuries, breakages, or spills to your laboratory instructor immediately. 8. Clean broken glass (glass that is not contaminated with any chemical reagents, blood, or bacteria) can be swept up using the dust pan and placed in the broken glass container. If the glass is contaminated in any way, keep the area clear to prevent tripping or laceration hazards, and consult your instructor for proper disposal guidance. A broken glass flow-chart is available in the lab to help you decide what to do. 9. Notify your instructor if any of the equipment is faulty. 10. Clean up your entire work area before leaving. Put away all equipment and supplies in their original places and dispose of reagents and infectious materials in the designated receptacles. Disinfect your work surface if the lab activity involved any 3 infectious materials. Otherwise, wipe the entire work surface down with a clean, wet paper towel (no soap). 11. Use the appropriate waste containers provided for any infectious or hazardous materials used in lab. 12. Safety information reagents used in the lab activities can be found in the Material Safety Data Sheets (MSDS), which are available in a binder in the lab. Know the location of the MSDS binder. We (faculty and students) should be fully aware of the properties of the reagents we are using. Please use the MSDSs. If you cannot find the MSDS for the reagent you are using in lab, inform your instructor. They are also relatively easy to find online. A Google keyword example is “Sodium Chloride MSDS.” 13. Use caution with the lab chairs. Because they are on casters, they can roll away when you are standing at your workstation. Make sure your chair is where you expect it to be before sitting down. Do not use your chair as a means of moving from one part of the lab to the other. 14. Wash your hands before leaving the lab room. 15. If class is held at an alternate location (e.g. a field trip), you will be expected to conduct yourself appropriately and follow lab safety rules where applicable. GENERAL OBJECTIVES OF THE COURSE At the end of the practical training in general, and experimental pharmacology the learner shall be able to: 1. List the various dosage forms and enumerate their advantages and disadvantages. 2. Advise patients about the proper use of medication devices, storage of medicines etc. 3. Retrieve drug information from appropriate sources. 4. Appreciate the role of good laboratory practice in promotion of rational diagnostics, therapy, and experimentation. 5. Realize the cardinal role of ethics in experimentation. 6. Order monitoring of drug levels where indicated and take appropriate remedial measures. 4 7. Prescribe rationally and in an individualized pattern. 8. Plan and carry out experiments to demonstrate the effect of drugs in experimental animals and isolated tissues. 9. Critically appraise drug advertisements. 10. Apply fundamental statistical tests to experimental data and interpret results. 5 LAB 1: STUDY OF DIFFERENT LABORATORY ANIMALS AND THEIR APPLICATION AIM: To study the advantages, disadvantages and experimental uses of different most commonly used laboratory animals. BACKGROUND INFORMATION: PHARMACOLOGY is the branch of science which deals with study of drugs on living systems. EXPERIMENTAL PHARMACOLOGY: deals with study of effect of various Pharmacological agents on different animal species. OBJECTIVES OF PHARMACOLOGY: ▫ To find out the therapeutic agent suitable for human use ▫ To study the toxicity of the drugs ▫ To study the mechanism and site of action of drugs LABORATORY ANIMALS: Animals those can be breaded and handled in laboratory. Examples: Rat, Mice, Guinea pig, Rabbits, Frogs, Cat, Dog, Monkey, Pigeon etc. 1. RATS (Rattus norvegicus ) • Albino rats of Wistar strain are commonly used • Other strains – ▫ Wistar kyoto rat ▫ Sprague Dawley rat ▫ Biobreeding (BBDP) rat ▫ Long-Evans rat ▫ Zucker rat ▫ Hairless rats (Rowett nude, Fuzzy, Shorn) ▫ RCS rats 6 2. MICE( Mus musculus) • Swiss albino mice are commonly used species • Other strains are – Balb/C and C-57 ADVANTAGES AND CHARACTERISTICS: ▫ Smallest ▫ Less drug required ▫ Easy to handle ▫ Cheap 3. GUINEA PIGS ( Cavia porcellus) ADVANTAGES AND CHARACTERISTICS: ▫ Docile animals ▫ Highly susceptible to TB and anaphylaxis ▫ Highly sensitive to histamine, penicillin ▫ Required exogenous vitamin C in diet 4. RABBITS (Lupas cuniculus) ADVANTAGES AND CHARACTERISTICS: ▫ Docile animal with large ears ▫ New Zealand white strains are widely used ▫ It has huge caceum and long appendix ▫ Enzyme atropine esterase is present in rabbit liver and plasma so it can tolerate large doses of belladona (atropine) 5. FROGS (Rana tigrina) ADVANTAGES AND CHARACTERISTICS: ▫ Used before 200 years ▫ Easily available during rainy season ▫ Amphibian animal and safe to handle ▫ Cannot breed in laboratory 7 LAB 2: STUDY OF DIFFERENT STAGES OF ANESTHESIA AIM: To study the different stages of anesthesia and calculate onset and duration of action of different anesthetic agent. ANIMALS: Wistar albino rats APPARATUS: Desiccator, Syringes, needle DRUGS AND SOLUTIONS: Diethyl ether, chloral hydrate BACKGROUND INFORMATION: General anesthesia was absent until the mid-1800’s. William Morton administered ether to a patient having a neck tumor, removed at the Massachusetts General Hospital, Boston, in October 1846. The discovery of the diethyl ether as general anesthesia was the result of a search for means of eliminating a patient’s pain perception and responses to painful stimuli. Anesthesia is defined as partial or complete loss of sensation with or without loss of consciousness as a result of disease, injury, or administration of an anesthetic agent, usually by injection or inhalation. STAGES OF ANESTHESIA:  STAGE 1 (ANALGESIA/ONSET/INDUCTION):  STAGE 2 (EXCITEMENT/DELIRIUM):  STAGE 3 (Surgical Anesthesia):  STAGE 4 (Impending Death/ Stage of Danger): PROCEDURE: Two Wistar albino rats will be weigh and keep in different cages. One rat will be injected by intraperitoneal route with chloral hydrate in a dose of 400 mg/kg and another rat will be inhaled by diethyl ether. Onset and duration of action of both anesthetic agents will be recorded. Above mentioned different stages of anesthesia will be observed. 8 RESULTS: S. No. Name of Anesthetic Onset of Duration of agent action Action CONCLUSION: 9