Practical Pharmacology - Aqaba Medical Sciences University PDF

Practical pharmacology Dr. Romany Helmy Thabet, M.D Assistant professor of pharmacology Aqaba Medical Sciences University 1  PRACTICAL LESSON (1) EXPERIMENTAL ANIMALS AND THEIR HANDLINGS Introduction Animals are widely used experimen...

Practical pharmacology Dr. Romany Helmy Thabet, M.D Assistant professor of pharmacology Aqaba Medical Sciences University 1  PRACTICAL LESSON (1) EXPERIMENTAL ANIMALS AND THEIR HANDLINGS Introduction Animals are widely used experimentally, especially in biological study in: a) Pharmacological research before testing drugs on human being. b) Practical pharmacology teaching to pharmaceutical and medical students as well as veterinary students. c) Practical physiology, animal lab sciences and anatomy teaching. Experimental animals should always be: a) Suitably bred. b) Gently handled. c) Properly used. Experimental animals are selected on the following bases: a) Nature of experiment. b) Type of drug activity to be evaluated. c) Specific character of experimental animals. Experimental animals are used for either in vitro or in vivo experiments: 1- “In vivo” experiments: in which we use whole intact animals and used for testing drug effects on: a) blood pressure, blood sugar b) autonomic drugs on eye c) CNS activity e.g., analgesic activity, psychotropic activity. d) acute and chronic toxicity studies. 2- In vitro experiments: in which we use only isolated organs from the animals (e.g., heart, intestine, skeletal muscles, uterus, trachea, etc.). o e.g., isolated rabbit intestine, isolated rabbit heart, rat stomach fundus, rat colon, frog rectus abdominus, rat uterus, guinea pig tracheal chain or some blood cells. o Used for testing drugs acting on ANS, CVS, skeletal muscles, autacoids and some hormones. The following animals are used in experimental pharmacology: 1- Large animals: (not commonly used): a- Monkeys c- Pigs b- Dogs d- Cats 2- Small animals: (commonly used): a) Mice c) Rats b) Guinea pigs d) Rabbits e) Frogs 2  1- Albino mouse (Swiss strain) Characteristics of albino mice: The smallest laboratory animals used. Weighing 25-30 g and easy to breed and maintain. Very sensitive to drug action. Consume small doses of drugs. Easily handled. Not used as a source of common isolated organs because of their small size. However, more recently mouse brain tissue are used in neuropharmacology for studying neurotransmitter receptor function. Drugs are best administered: - Intraperitoneal injection (I.P.) into mouse peritoneal cavity. - Intravenous injection (I.V.) into mouse superficial tail vein. - Oral by stomach tube. Scientific uses of mice in research: a) In cancer research. b) In genetic research. c) In toxicity studies (LD50 determination). d) In screening of drugs acting on CNS as analgesics. Proper method of mice handling: - The mouse is lifted from the tail with the right hand. Animal allowed to grip on wire mesh of the cage with its fore legs. Then the animal is grasped from the nap of the neck between the thumb and index finger using left hand. Finally, the tail is then transferred from the right hand to the left hand between thumb and small fingers. Discussion: Advantages of mice: ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ Proper methods of drug injection into mice: ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ Is it possible to obtain isolated organ preparations from mice – why? ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ 3  2. Albino rat (Rats) Albino rats (Wistar strain) are different from mice and characterized by: Larger weight than mice (200-500 g); easily to breed and maintain. Withstand long period of experimentation under anesthesia. Easily handled if treated kindly. Drugs injected Iintravenously (I.V.) into their tail veins, subcutaneously (S.C.), Intramuscularly (I.M.) or intraperitonealy (I.P.). Also, drugs can be administered orally into rats by means of stomach tube. Rats can be used as a source of isolated organ preparations as rat stomach fundus, rat colon and rat uterus in addition to the rat brain tissues used extensively in radioreceptor ligand studies. Rats do not have vomiting centers and hence not possible to use them in the screening of antiemetics. The other strains of rats are: Sprague –Dawley and Porton. Uses of rats in research: a) In acute and chronic toxicity studies. b) Testing of drugs acting on CNS particularly psychotropic drugs because rats can be trained well to perform skillful tasks. Proper method of handling of rats: Rats can be handled easily or difficulty based on manner of treatment. The rats are handled as the following: a) The rat is grasped from its tail, placed on a hard surface and the left hand is slipped over the rat’s back and is moved toward the head. b) The right front paw of the rat is restrained between the base of the thumb and the index finger and the body of the rat is kept tight by holding the tail. Discussion: Q: What are the advantages of rats over mice? -------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------- 4  Q: Appropriate methods of drug administration into albino rats: -------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------- Q: Mention different isolated organ preparations that can be obtained from albino rats. -------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------- 3. Guinea pigs Guinea pigs are characterized by the following: Weight (400-600 g) Very docile and easy to raise and maintain Highly sensitive to histamine and so commonly used for screening of histamine-like and antihistaminic drugs. Useful in experimental asthma to study bronchodilators. Also, they are useful in studying local anesthetics, and some microbial models as amoebiasis and cholera since they are very sensitive to infections by these microorganisms. Can not synthesize vit C and this vitamin should be supplied in their diet. Can be used as a source isolated muscle preparations e.g., guinea pig tracheal chain, guinea pig vas deferens, guinea pig ileum. Very sensitive and tame animals. Proper method of handling of guinea pig: The animal is hold easily by one hand making the animal close to the body. Q: What are the main charcters of guinea pigs? ------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------ 5  Q: Appropriate methods of drug administration into guinea pigs: ------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------- Q: Mention different isolated organ preparations that can be obtained from guinea pigs. ------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------- 4) Rabbits Characteristics: Rabbits are very docile animals. Easily injected intravenously into marginal ear veins. Drugs administered orally into rabbits by stomach tube. One peculiar thing about rabbits is that they contain atropinesterase enzyme which cause atropine resistance. Use of rabbits in research: Generally used for pyrogen tests. To study the effect of drugs on the eye, CVS, ANS, and neuromuscular junction (NMJ). As a source of isolated organ preparations: e.g., rabbit intestine, Langendorff’s preparation (isolated rabbit’s heart) and rabbit aortic strips. Proper method of rabbit’s handling: Rabbits should never be lifted from their ears because this will cause pain to animal giving utter sound. Rabbits are handled by grasping with left hand from their loose skin at the nape of the neck. The buttocks are placed on the right hand and the animal is made closer to the body of the experimenter. Q: What are the peculiar characters of rabbits and its clinical significance? ------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------ 6 Q: Appropriate methods of drug administration into rabbits: ------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------- Q: Mention different isolated organ preparations that can be obtained from rabbits. ------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------- Q: Mention the different uses of rabbits in the research? ------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------- 5. Frogs Characteristics: Frogs are amphibian (cold-blooded animals). Frogs must be kept moistened with water to absorb water through their skin. Usually drugs administered into frogs subcutaneously (S.C.) in their dorsal lymph sacs. Isolated frog heart, rectus abdominus, sciatic nerve preparation are some of the isolated organs obtained from frogs. Uses of frogs in research: Frog rectus abdominus and sciatic nerve muscle preparations are commonly used in the screening of drugs acting on skeletal muscle as neuro muscular blockers. Also, used for screening of drugs on CVS. Proper method of frog handling The frog is taken by the left hand and is held by grasping its trunk. The head of the frog is held between the thumb and index fingers. The hind limbs are placed between the fourth and fifth fingers. Discussion: Q: What is the most appropriate parenteral route of drug administration into amphibian? ---------------------------------------------------------------------------------------------------------------------------- ---------------------------------------------------------------------------------------------------------------------------- ---------------------------------------------------------------------------------------------------------------------------- 7 What Are the Alternatives to Experimental Animals? Ideally these should maintain the benefits & reduce the costs: o Computer-based simulations √ o Video and interactive video √ o Mannequins (Kokar rat, microsurgery), models and simulators o Non-animal experiments (e.g., using plant tissues, post-mortem materials, cultured cells). o Clinical cases (PBL) √ What are computer simulations of the preparations? use models to generate tissue responses allow control over experimental parameters - good for design highly flexible need direct/indirect tutor support Examples: Guinea pig ileum, Cardiolab, Vascular Rings, Sciatic nerve-anterior tibialis, Frog Heart, Intestinal Absorption, Cat Nictitating Membrane, Langendorff Heart, rat Blood Pressure, Inflammation Pharmacology, Finkleman, Respiratory Pharmacology, Intestinal Motility, Renal Function Do simulations work properly? Yes, some of the evidence addressed some learning objectives as: knowledge gain is equivalent. Data handling skills. Experimental design skills. Communication skills. Group work. Costs are less. Promoting staff-student interaction. Better support for weaker students. 8  PRACTICAL LESSON (2) ROUTES OF DRUG ADMINISTRATION For a drug to be effective it has to attain certain concentration at its site of action. For this reason, drugs are applied or administered by numerous channels or avenues. The selection of the route of administration depends on the drug itself, the pharmaceutical preparation in which the drug is prescribed and the state of the patient. The principal methods of giving drugs include: 1- Oral administration: The drug is introduced through the mouth in a variety of dosage forms including solutions, emulsions, suspensions, powders, cachets, tablets and capsules. 2- Parenteral administration: This includes all forms of injections either of solutions or suspensions and includes: a) Subcutaneous injection. b) Intramuscular injection. c) Intravenous injection. d) Intrathecal injection. e) Intraperitoneal injection. 3- Inhalation administration: Vapors of volatile compounds, gases and aerosols may be readly administered by inhalation through suitable devices. 4- Topical administration: The drug is applied either for systemic action or more commonly for a localized action. Drugs used for this purpose are dispensed in the form of powder, solution, lotion, cream, liniment and ointment. 9 5- Mucosal administration: The mucosa provides an excellent method of administration of many drugs. This includes: a) Sublingual: By inserting a tablet under the tongue. b) Rectal: Suppositories or solutions are used for this purpose : c) Nasal: Spray of solutions are used for this purpose. d) Vaginal: Solid preparations (pessaries) or lavages are available for this purpose. Each of these routes has its particular advantages and certain shortcomings. DISCUSSION: Get yourself familiar with the prominent advantages and disadvantages of each of these routes. Mention some other channels of drug administration not mentioned in this text. a) -------------------------------------- d) ---------------------------------- b) -------------------------------------- e) ---------------------------------- c) -------------------------------------- f) ---------------------------------- COMMENTS Advantages of oral route: ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ Disadvantages of oral routes: ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ 10 Mention advantages and disadvantages for other 3 routes of administration: ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ The quantitative response to a drug depends largely upon its avenue of entrance to the body. The qualitative response to a drug also may be modified by the avenue of administration. OBJECTIVES 1- To get acquainted with the different channels by which drugs can be applied or introduced to the body. 2- To be familiar with the technics of applying these channels 3- To appreciate some of the factors which may influence drug action when applying these channels. 4- To recognize some advantages and disadvantages of each of these channels. EXPERIMENT 2-a Solutions of certain drugs will be available for administration by different routes. Notice the proper handling and the technic used for administering the drug. 11 Before drug administration, record normal respiration, and check it at intervals after giving the drug. Administer 30 mg/kg of pentobarbital sodium to rats by: a) Oral. b) Intraperitoneal (I.P.) c) Intramuscular (I.M.) Note the time of administration and record the time elapsing between administration and the following events : - Loss of righting reflex. - Ataxia. - Anesthesia. Also, record the onset and duration of anesthesia by noting the time of appearance and disappearance of these events. RESULTS AND CONCLUSIONS ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ 12 ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ EXPERIMENT 2-b A drug will be available for administration by both oral and parenteral routes. Record respiratory pattern, CNS and GIT activity before and after administration. Administer magnesium sulfate to two rats as follows: a) I.P. 1.0 gm/kg b) Oral 5.0 gm/kg using a stomach tube. RESULTS AND CONCLUSIONS ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ 13 DISCUSSION (Q) Try to figure out certain factors which may influence the rate of absorption of drugs from different sites of administration and discuss their clinical significance with your instructor. (Q) Try to mention certain drug examples and their suitable methods of administration. COMMENTS ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ 14  PRACTICAL LESSON (3) THE LIVER MICROSOMAL DRUG METABOLIZING SYSTEM Background - Biotransformation is a major mechanism for drug elimination through phase I or phase II reactions. Metabolism is catalyzed by specific enzyme systems in the liver; called liver microsomal enzyme systems. Biotransformation process influences the activity and the biological half-life of a drug. - Biotransformation may result in: Metabolites that are more polar (usually inactive metabolites) than the parent drug. Therefore, biotransformation usually terminates the pharmacologic action of the parent drug. Metabolites that may be more active or more toxic (Ocassionally) than parent drugs. - Although, most drug metabolites are less biologically active than their parent compounds (detoxification), in some cases a more toxic metabolite is produced (lethal synthesis). In this latter case the administered drug is really only a "pro-drug" which is biotransformed into a pharmacologically active metabolite. Similarly, more toxic metabolites may be formed as a result of biotransformation e.g., paracetamol large dose. - The duration and intensity of action of many drugs in animals depend on the activity of drug-metabolizing enzyme systems localized in the microsomal fraction of liver homogenates. - During the last few decades, we became aware that some environmental factors may play an important role in regulating the activity of these enzyme systems. Dietary and nutritional factors, X-ray irradiation, hormonal changes in the body, and ingestion of some foreign chemicals (including drugs) are some of the factors which may influence the activity of these microsomal enzyme systems. 15 - Chronic administration of compounds that stimulate the activity of drug- metabolizing enzymes in the liver microsomes markedly decreases the duration of action of a subsequent dose of the same agent or some other agents. Examples includes drugs such as diphenylhydantoin, rifampicin and several barbiturates. Similarly, other environmental agents are known to stimulate the activity of drug-metabolizing enzymes in the liver microsomes as insecticides, polycyclic hydrocarbon in cigarette smoke, food preservatives and some dyes. - On the other hand, few are known to depress the microsomal oxidative system on chronic long-term use. This has been shown with ketoconazole, chloramphenicol, erythromycin, cimetidine, allopurinol, disulfiram in addition to starvation. Enzyme induction and enzyme inhibition represent the major sites of drug-interactions. Q: Discuss factors affecting biotransformation. ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ Q: Mention phases of metabolic reactions. ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ 16 EXPERIMENTAL: OBJECTIVES After completion of the session, students will be able to: 1- Identify the role of microsomal enzyme system in drug biotransformation and disposition. 2- Discuss the phenomena of enzyme induction and enzyme inhibition and their clinical significance in drug-interactions. 3- Recognize the influence of the nutritional state particularly starvation on microsomal enzyme system activity and consequently drug action and safety. EXPERIMENT 3: Effect of phenobarbital and starvation on the sleeping time of thiopental in mice Three male mice will be supplied to each group of students. The first is a normal untreated animal, the second has been treated with phenobrabitone 20 mg/kg i.p. twice daily for one week prior to the experiment and the third was starved for 36 hours. During the 8th day, inject each mouse with 100 mg/kg thiopental i.p.. Observation: Record the onset of time of loss of righting reflex and the duration of this hypnotic effect. Repeat the experiment more than one time RESULTS AND CONCLUSIONS Results Mouse number Average Onset of Average duration of righting reflex hypnosis Mouse 1 Mouse 2 Mouse 3 17 COMMMENTS ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ CONCLUSION: ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ DISCUSSION (Q) Mention some examples of pro-drugs and their active metabolites. ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ (Q) Mention the names of some agents or conditions of enzyme-inducers ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ 18 (Q) List a story with some instances where enzyme induction or enzyme inhibition may lead to drastic drug action and/or disasters. ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ (Q) Name some agents or conditions which are known to inhibit the microsomal enzyme system. ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ CONCLUSIONS ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ 19  PRACTICAL LESSON (4) AUTONOMIC DRUGS AND THE EYE Figure (1): Anatomy of the eye & autonomic receptors. Background: The internal muscles of the eye are all controlled by the autonomic nervous system. The iris is controlled by two reciprocating sets of muscle fibers, the circular sphincter with oculomotor innervation (parasympathetic) and the radial dilators innervated from the superior cervical ganglion (sympathetic). Autonomic drugs affecting pupil size, accommodation, intraocular pressure, and other parameters. Effects of autonomic drugs on the pupil size: Iris muscles controlling pupil size: a) Constrictor pupillae muscles (circular muscles): innervated by parasympathetic nerve and contains muscarinic (M) receptors. Muscarinic agonists e.g., pilocarpine causes contraction of circular muscles leading to miosis (decrease pupil size). In contrast, muscarinic antagonists e.g., 20  atropine or homatropine cause paralysis of constrictor pupillae muscles leading to predominance of radial muscles of the iris with the results of passive mydriasis (with loss of light reflex). b) Dilator pupillae muscles (radial muscles of the iris): innervated by sympathetic nerves and contains α1 receptors. Adrenergic α1 receptor agonists e.g., phenyephrine produces active mydriasis (increase pupil size). In contrast, adrenolytic α1 receptor blockers e.g., prazosin or phentolamine produces miosis. Effects of autonomic drugs on accommodation:  Accommodation means ability of the eye to see near and far objectives through alteration in refractive power of the lenses. Only ciliary muscles are responsible for accommodation. Ciliary muscles activity is controlled by parasympathetic nerve and contains M receptors. - Muscarinic agonist → contraction of ciliary muscles → relaxation of suspensory ligaments → lens becomes more convex → spasm of accommodation (cyclospasm; fixation of eye to near vision). - Muscarinic blockers → paralysis of ciliary muscles → stretching of suspensory ligaments →lens becomes more flat → paralysis of accommodation (cycloplegia; fixation of eye to see far objectives only).  All sympathetic drugs (agonist and antagonists) have no effect on accommodation because there is no sympathetic innervation for ciliary muscles. Effects of autonomic drugs on intraocular pressure (IOP): The aqueous humor which carries nutrients for the non-vascular structures of the eye is a filtrate from the plasma in the capillaries of the ciliary body and posterior surface of the iris. The fluid is produced under pressure so that the normal intraocular tension (I.O.P.) in man is 12-20 mm. Hg. It is obvious that the production of aqueous humor must be balanced by an equal loss (drainage) from 21 the eye. The aqueous humor is drained through the canal of Schlemm at the iridocorneal angle. 1- Effect of parasympathomimetics: e.g., pilocarpine produces initial transient increase in IOP (due to VD that causes increases in aqueous humor formation followed by persistent decrease in IOP (due to improvement of drainage by two mechanisms: a) In close angle glaucoma: circular muscle contraction (Miosis) removing physical obstruction and so increase drainage of aqueous humor. b) In open angle glaucoma: Contraction of ciliary muscles with consequent opening of trabecular meshwork pores and hence increases drainage of aqueous humor. 2- Effect of parasympatholytics: e.g., Atropine causes increase in IOP by obstruction of drainage of aqueous humor due to relaxation of circular muscles & passive mydriasis and so may worsen close angle glaucoma. Similarly, in open angle glaucoma, relaxation of ciliary muscles decreases drainage of aqueous humor by trabecular mesh work pores and so worsen the case. Therefore, it is recommended that all parasympatholytics are contraindicated almost in all types of glaucoma (both open and close angle types). 3- Effect of sympathomimetics: e.g., epinephrine. Due to its mydriatic effects, it worsens physical obstruction in close angle glaucoma with a resultant increase in IOP. However, in open angle glaucoma, epinephrine and allied drugs decrease production of aqueous humor by its vasoconstrictor effect with consequent decrease in IOP. Therefore, sympathomimetics are useful in open angle glaucoma but contraindicated in close angle glaucoma. 22 Effects of autonomic drugs on the light reflex:  Light reflex means Shining light to one eye will cause miosis in both eyes to protect eyes against light.  This reflex needs healthy unblocked constrictor pupillae muscles.  So, muscarinic anatgonists e.g., atropine → paralysis of constrictor pupillae muscles → loss of light reflex.  Therefore, light reflex is preserved with all autonomic drugs EXCEPT muscarinic antagonists.  Light reflex differentiates between passive and active mydriasis. Effects autonomic drugs on lacrimation:  Parasympathomimetics increase lacrimation markedly (++++) due to stimulation of muscarinic receptors in lacrimal glands. However, parasympatholytics decrease lacrimation markedly causing drynes of the eye or sandy eyes). EXPERIMENTAL OBJECTIVES: After completion of the session, students will be able to: 1- Recognize the mechanism of different autonomic drugs on the eye. 2- Identify the different changes that can be produced by drugs acting on the eye. 3- Select properly various drugs in patients with glaucoma. 23 EXPERIMENT 1- EFEECTS OF PARASYMPATHETIC DRUGS ON THE EYE: Instill into the right eye of a rabbit 2 drops of the physostigmine solution and into the left eye 3 drops of the pilocarpine solution. Record changes in pupillary size at repeated intervals and test for light reflex. Once miosis is fully developed in both eyes instill 2 drops of homatropine solution and observe the effect. After the effect is shown add to the right eye 2 drops of physostigmine solution and to the left eye 3 drops of pilocarpine solution and record your observation. RESULTS EFFECT OF PARASYMPATHETIC DRUGS ON THE EYE Treatment Ocular Manifestation Pupillary size Light reflex Right eye : Before treatment Physostigmine Homatropine Physostigmine Left eye : Before treatment Bilocarpine Homatropine Pilocarpine 24  2- Repeat the same experiments using homatropine on the left eye and phenylephrine in the right eye and record the results. Results: Treatment Ocular Manifestation Pupillary size Light reflex Left eye : Before treatment Homatropine Right eye : Before treatment Phenylephrine DISCUSSION (Q) Outline the autonomic receptors in the eye and drug acting on each receptor. ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ 25 ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ (Q) Mention the clinical applications of homatropine and physostigmine in the field of ophthalmology. ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------ (Q) What are the differences between: A. Active and passive mydriasis? ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ B. spasm and paralysis of accommodation? ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ COMMENTS ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ 26  PRACTICAL LESSON (5) EFFECTS OF DRUGS ON ISOLATED TOAD HEART (LANGENDORFF’S) Figure (2): Autonomic innervations and receptors on the heart Background: - Heart is innervated by both sympathetic and parasympathetic "vagus" nerves. Therefore, the heart contains both adrenergic and cholinergic receptors (β1, M2 and N) receptors respectively in addition to H2 receptors. - Drugs may influence the rate (chronotropy) and force of contraction (inotropy) of the heart. An increase in the heart rate is called “positive chronotropic” effect and a decrease in heart rate is called a “negative chronotropic” response. Similarly, an increase in the force of contraction is called “positive inotropic” response and a decrease in force of myocardial contraction is called “negative inotropic” response. Sympathomimetic such as adrenaline, noradrenaline, isoprenaline and dobutamine produce both positive inotropic and positive chronotropic responses. This means that these drugs increase both contraction and heart rate (tachycardia) respectively. On the other hand, parasympathomimetics like acetylcholine, pilocarpine and physostigmine produce negative inotropics and negative chronotropic effects. 27  OBJECTIVES: To demonstrate the effect of different drugs either alone or combined with different blockers on the isolated toad’s heart. Requirements: Frogs, frog Ringer physiological salt solution, frog board, surgical instruments universal heart lever (Langendorff’s apparatus). Experiments Try to demonstrate the effect of drugs as follows: ♦ Sympathomimetics (stimulatory) a− Adrenaline. b− Noradrenaline. C- Dobutamine. In each case try the drug alone or with antagonist. ♦ Parasympathomimetics (Inhibitory): a− Acetylcholine. b − Methacholine c − Nicotine. ♦ Cardiac glycosides (stimulatory): e.g., Digoxin. ♦ Other agents: e.g., Histamine and verapamil (inhibitory). ♦ Ions : a − Calcium (stimulatory. b − Magnesium (inhibitory). Procedure: 1. Pith the frog and pin it to the frog board. 2. Give a midline incision in the abdomen, remove the pectoral girdle and expose the heart. 3. Carefully remove the pericardium and put a few drops of frog Ringer over the heart. 4. Trace the inferior vena cava, put a thread around it and connect venous cannula with perfusion bottle containing frog Ringer at room temperature. Insert the cannula in the vein well and tie to assure the cannula in its place. 5. Make a small cut in the aorta for perfusate to come out. 28  6. Pass a thin pin hook through the tip of the ventricle, and with the help of a fine thread attached to the hook, tie it to the free limb of universal lever. Adjust proper tension and magnification by altering the height of the lever. Record the normal contraction of the heart on smoked drum or recorder. Inject drugs under investigation in various concentration up to maximum 1 ml with keeping at least 5 min intervals between doses addition. Figure (3): Simple apparatus used to perfuse isolated toad’s heart. Experiment 5-1 Figure (4): Effect of histamine with and without pretreatment by cimetidine 29  Comments: ------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------- Experiment 5-2 Figure (5 ): Effect of acetylcholine with and without pretreatment by atropine Comments: ------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------- 30 Experiment 5-3: Figure (6 ): Effect of adrenaline with and without pretreatment by propranolol COMMENTS: ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------ Discussion: Q: What do you understand by the terms tachycardia and bradycardia? ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ----------------------------------------------------------------------------------------------- 31 Q: What do you mean by the terms cardiac stimulants and depressants? ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ----------------------------------------------------------------------------------------------- Q: What types of autonomic receptors located in the heart and their functions? ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ----------------------------------------------------------------------------------------------- Q: Mention the cardiac stimulants and explain their mechanisms of actions. ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ----------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ----------------------------------------------------------------------------------------------- Q: Mention the cardiac depressants and explain their mechanisms of actions. ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ----------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ----------------------------------------------------------------------------------------------- 32 Q: What are the therapeutic benefits of cardiac stimulants? ------------------------------------------------------------------------------------------------ ----------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ----------------------------------------------------------------------------------------------- Q: What are the therapeutic benefits of cardiac depressants? ------------------------------------------------------------------------------------------------ ----------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ Conclusion: ------------------------------------------------------------------------------------------------ ----------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ----------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ----------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ----------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ 33  PRACTICAL LESSON (6) EFFECTS OF DRUGS ON THE ISOLATED INTESTINE Background: The isolated intestine preparation is one of the classical preparations in physiology and pharmacology for bioassays, or the study of drug action and autonomic control of motility of smooth muscles of the GIT. The classic way of in vitro observing the intestinal motility is to suspend a piece of small intestine (from a rabbit or a guinea pig) vertically in an organ bath between an aeration tube and a recording lever. Physiology of intestinal smooth muscle contraction: Visceral smooth muscle contraction occurs spontaneously, meaning that muscle cell action potentials are generated without input from either the motor or autonomic nervous system. The muscle cells undergo rhythmic oscillations i n membrane potential which, occasionally, reach the threshold of an action potential, and, thus, generate a spike. The action potential spreads via gap junctions from muscle cell to muscle cell, initiating a wave of muscle contraction in its wake. Visceral smooth muscle cells also exhibit muscle tonus, a state of long-term, steady contraction. The tonus is variable, depending on the number of muscle cells that participate. Motility in the small intestine, as in all parts of the digestive tube, is controlled predominantly by excitatory and inhibitory signals from the enteric nervous system. This motility is however modulated by inputs from the central nervous system (sympathetic nerve, parasympathetic nerve). The rhythmicity and tonus inherent in the intestinal smooth muscle may be enhanced or suppressed by two nerve plexuses found between the layers of muscle and mucosa (Auerbach's and Meissner's plexuses). Known as the enteric nervous system of the gut, the activity of the plexuses can be modified by the autonomic nervous system. When a piece of intestine i s removed for study. The plexuses remain viable and can be stimulated or inhibited using parasympathomimetic or sympathomimetic drugs (mimicking the action of the parasympathetic and sympathetic nervous systems respectively).The autonomic control of the gut i s very complicated. 34 There are alpha- and beta-adrenergic receptors on the smooth muscle cells which will respond to epinephrine and norepinephrine. Both alpha and beta receptors inhibit muscle tone and rhythmicity. The parasympathetic control of the gut is via cholinergic preganglionic fibers which synapse with enteric neurons. When activated, the parasympathetic fibers release acetylcholine at the enteric neurons, thus stimulating the enteric neurons (post ganglionic NN receptors) to release acetylcholine at their neuro-effector junctions. Enhanced smooth muscle tone and rhythmicity result from actions of acetylcholine on M3 receptors on smooth muscles. A number of gastrointestinal hormones (Gastrin, Cholecystokinin) or other mediators (Histamine, prostaglandins and peptides) appear to affect intestinal motility to some degree. The stimulatory effect of Barium chloride on smooth muscles of intestine will be discussed. Preparation of isolated segments of the rabbit i intestine: The rabbit should be starved f o r 24 hours prior to use. Food in the gut will result in a messy dissection and will necessitate flushing to remove the gut contents, a practice which could damage the intestine. Before sacrificing the rabbit: 1. Prepare Tyrode's Ringer solution and place about 250 mL of this solution in a flask. Tyrode's Ringer Solution: NaCl 8.0 g KCl 0.2 g NaHCO3 1.0 g MgCl2.6 H20 0.1 g CaCl2.2 H20* 0.1 g NaH2PO4.H2O 0.05 g Dextrose 1.0 g * Dissolve the calcium chloride separately and add last Bring up to 1 L with distilled water. 2. Cut sections of thread (or 4-0 silk) in o 5 cm lengths. One piece will be needed for each segment of gut. 35 3. You will need a pair of fine (iris) scissors, coarse scissors, and curved forceps. Hemostats may also be helpful in retracting the abdominal muscles. 4. A large Petri plate or other container for holding the segments of gut will also be necessary. 5. Wash all instruments and glassware before use. Also make sure your hands are clean before handling the intestine. Sacrificing the rabbit and removing the intestinal segments: 1. Since anesthetics can affect motility of the gut, the animal should be sacrificed by cervical dislocation, without use of anesthetic. 2. Shave the abdomen and make a mid- line incision through the skin and abdominal muscle using a pair of coarse scissors. 3. Locate the ileum, a section located near the end of the small intestine. Select a 3-cm segment of the ileum that is supplied by seen blood vessels in the mesentery. Sever the gut and remove this segment along with the mesentery containing the vessels and nerve. Place in a Petri dish containing cold Tyrode's Ringer solution. N.B.: If the rabbit has been starved f o r 24 hours, the gut segment should be free of chyme. However, if chyme is present, it can be removed by carefully flushing the segment with Tyrode's. A syringe filled with Tyrode's may be placed inside one end of the segment, and using very little pressure; the contents can be carefully forced out into a waste container. Caution must be exercised because high pressure will severely damage the tissue. 4. Repeat this procedure until enough segments have been removed to supply your laboratory needs f or the day. I f the extra pieces of ileum are kept i n ice cold Tyrode's; they will stay viable f o r several hours. Do not store i n warm Tyrode's. 36  Figure (7): Isolated rabbit intestine immersed in Tyrode’s solution at 37 C. By using different drugs that bind to the receptors on intestinal smooth muscular cells, we can observe the actions of drugs and analyze their action mechanism. Suggestions: 1. The ileal segments will shorten when placed into the cold Tyrode's solution and motility will cease. However, when mounted in the warm (37°C) Tyrode's solution inside the muscle bath, motility will return within 5-10 min. 2. As the muscle warms i n the bath, it will relax and segmental and peristaltic waves will be evident. 3. It is essential that the muscle baths be a t 37°C at the start of class. This means starting the circulation of warm water through the baths at least 1/2 hour before class. Also, it is essential that the extra Tyrode's solution for the muscle baths be at 37°C. 4. I t is not necessary to warm the drug solutions used i n the experiment since they will be added directly to the muscle bath in very small amounts (0.1 to 1.0 mL of drug/100 mL of Tyrode's). 5. One of the advantages in adding drug directly to the bath in concentrated form i s that rapid changes i n muscle tonus can be clearly recorded. We recommend that the Tyrode's solution be replaced between drug additions so as not to disturb the motility directly before the application of the drug. It may take 1-2 minutes for the rhythm to stabilize after replacing the solution i n the muscle bath. 6. Fix the specimen (1-1.5 cm) in the isolated organ bath. The specimens are mounted vertically, one end is connected to the lower hook of the bath and the 37 other end is connected to a force transducer. Tyrode’s solution, 10 ml (37ºC), is added into the bath. The specimens are allowed to equilibrate at 2-3 g tension for 30 min. 7. Observe the rhythmic contraction and tension level of the intestinal muscle; trace out the normal contraction curve until the specimen becomes stable. The isolated intestinal tract from many species of animal can maintain active rhythmic movements for a long time under suitable conditions. Experiment 6-a: Effects of stimulants and their blockers on isolated rabbit intestine: In this experiment, the effects of BaCL2 acetylcholine and histamine on the motility of isolated ileum are investigated. Atropine and chlorpheniramine (H1 antagonist) are used as tool drugs to analyze the underlying mechanisms. Aims: 1. to observe the effect of acetylcholine, histamine, BaCl2 and their blockers atropine and chlorpheniramine and MgCl2 on smooth muscle of ileum and analyze the mechanism of action of these stimulants on intestine. 2. to grasp how to do experiment in vitro. 38 Principles Ach is a muscarinic receptor agonist which stimulates the contractions of intestinal smooth muscle and increases its tension, while atropine is a muscarinic antagonist, so it can antagonize the effect of Ach. Histamine activates H1 receptors on intestinal smooth muscle while chlorpheniramine antagonize these actions. Materials 1-Prepare small intestine from a rabbit (discussed before) 2-Drugs: A) - 1:100000 acetylcholine chloride (ACh) B) - 0.1% atropine sulfate C) - 1:100000 histamine phosphate D) - 1:1000000 chlorpheniramine E)- 1% barium chloride (BaCl2) 3-Three conditions that an isolated organ to be alive in vitro: （1） Temperature: 37 ℃ （2） Gas: air is ok to ileum, no need for oxygen. （3） Nutrient solution: Tyrode’s solution Procedure: 1- Add a drop of ACh into the bath solution and observe for 2-3 min, record the peak of the change of the contractive tension, and then wash off. 2- Add a drop of ACh into the bath solution. When the contraction of intestinal smooth muscle reaches the peak, add a drop of Atropine. After the contraction of intestinal smooth muscle becomes stable, record the tension and then wash off. 3- Add a drop of Histamine into the bath solution, observe and record the peak of the change of the contractive tension, and then wash off. Add a drop of Chlorpheniramine into the bath solution and then add a drop of Histamine. Record the tension of the contraction peak and then wash off. 4- Add a drop of BaCl2 into the bath solution. When the contraction of intestinal smooth muscle reaches the peak, record the tension and then add a drop of Atropine. After the contraction of intestinal smooth 39  muscle becomes stable, record the tension and then wash off. The figure shows the effects on specific computer software. Results …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… Discussion …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… Conclusion …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… 40 Experiment 6-b: Effects of inhibitory agents on the isolated intestine using physiography Principle: Both alpha- and beta-adrenergic agonists can inhibit smooth muscles of the intestine. The figure here are obtained from physiography (not from a computer software) Figure (8-A): effects of Atropine on acetylcholine stimulant action Figure (8-B): Norepinephrine (α and β agonist) and isoproterenol (β agonist) decreases intestinal motility Figure (8-C): Ach increases frequency and tone of contractions 41  Figure (9): Stimulatory effect of acetylcholine and inhibitory effect of adrenaline Discussion: …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… Conclusion: …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… The following table shows common drugs that affect intestinal motility Stimulatory drugs Inhibitory drugs Parasympathomimetics Symapathomimetics BaCl2 MgCl2 Histamine, Serotonin Nitrates, methylxanthines Papverine, morphine Questions: What are the possible therapeutic uses of intestinal stimulants? …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… What are the possible therapeutic uses of intestinal relaxants? 42  PRACTICAL LESSON (7) EFFECTS OF DRUGS ON THE BLOOD PRESSURE IN THE ANESTHETIZED RABBITS 1. Objective and Principle: Study the equipment and methods for acutely recording the blood pressure of anesthetized rabbit. Observe the effects of drugs which act on the efferent peripheral nervous system on the blood pressure of anesthetized rabbit and drug interaction. 2. Experimental and Material: A rabbit (2~3 kg), operating table, surgical instruments, force-displacement transducer, titer tube, syringes, heparin solution, normal saline, 25％ urethane solution are used. Drugs used in this experiment may be found afterward in detail. 3. Method and Procedure: 1- A rabbit is weighed and anesthetized by 25％ urethane solution (3.5ml/kg), and then fixed on the surgical table. 2 - Surgery. Cut the fur on the front of neck and the skin along midline. Isolate right carotid artery and insert an arterial cannula filled with anticoagulant and connect to force displacement transducer and either a software system or a physiograph. Don’t get out the artery forceps until the force displacement transducer is well connected. Inject (i.v.) continuously normal saline from ear edge vein (marginal ear vein). 3 - Administration and observations. Drugs are given one by one as following. After each administration, inject 2mL normal saline for washing. Don’t give next drug until the blood pressure restore to pre-administration or stable level. A. Observe the effects of adrenergic agonists on blood pressure: a. adrenaline. b. noradrenaline. c. isoprenaline. B. Observe the effects of α adrenoceptor blocking drug on the action of adrenergic drugs: a. Effect of phentolamine alone. b. Effect of phentolamine on adrenaline. c. Effect of phentolamine on noradrenaline. d phentolamine, isoprenaline. 43 C. Observe the effects of β adrenoceptor blocking drug on the action of adrenergic drugs: a. Effect of propranolol on adrenaline. a. Effect of propranolol on noradrenaline. c. Effect of propranolol on isoprenaline. D. Observe the effects of cholinergic drugs on the blood pressure. a. acetylcholine. b. Effect of atropine on acetylcholine Table 3: Effects of drugs on blood pressure of anesthetized rabbit Drug (s) Effect on blood pressure Explanation Adrenaline Noradrenaline Isoprenaline (Isoproterenol) Phentolamine alone Adrenaline after phentolamine Noradrenaline after phentolamine Isoproterenol after Phentolamine Adrenaline after propranolol Noradrenaline after propranolol Isoproterenol after Propranolol Acetylcholine Acetylcholine after atropine Questions: 1-Discuss briefly the interaction mentioned in the above table …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… 44 …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… 2-Discuss the similarities and differences of cardiovascular effects among adrenaline, noradrenaline, and isoproterenol. …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………… 3-Explain epinephrine reversal …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… 45 Figure (10): Effects of adrenergic agonists on blood pressure and heart rate Discuss the differences between adrenaline and noradrenaline effects on heaart rate-explain. …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… Figure (11): Effects of adrenergic antagonists on biphasic response of epinephrine 46 Figure (12): Diagram showing biphasic response of epinephrine. 47  CLINICAL CASE GLAUCOMA Physiology of IOP - Normal IOP: 12-20 mmHg. - IOP above 20 mmHG is abnormal = glaucoma Risk factors of glaucoma: - Sex (women) - Diabetes - Family history - Age (over 40) - Eye injuries and eye disease. - Steroidal use - Migraine - Race: Black race (4-6 times more) Anatomy of glaucoma Clear liquid called aqueous humor circulates inside the front portion of the eye. To maintain a healthy level of pressure within the eye, a small amount of aqueous humor is produced constantly, while an equal amount flows out of the eye through a microscopic drainage system—the trabecular meshwork. If there is a defect in drainage system or overproduction in aqueous humor, glaucoma occurs. Functions of Aqueous humor: - Maintains the intraocular pressure of the eye within norma;l range (10-20 mm Hg). - Provides nutrition (e.g., amino acids and glucose) for the avascular ocular tissues; posterior cornea, trabecular meshwork, lens, and anterior vitreous. - Acts as an antioxidant through ascorbate. - Protect the eye against dust, wind, pollen grains and some pathogens. 48 Pathophysiology of glaucoma: Glaucoma is a chronic long- life eye disease. One of the leading causes of blindness. ↓ drainage of aqueous humor through trabecular meshwork ⇒ ↑ IOP inside the eye ⇒ damage optic nerve ⇒ Irreversible blindness. In glaucoma: Rise in IOP→ visual damage or visual field loss. Optic nerve damage irreversibly (Cupping of Optic Nerve). Diagnosis of glaucoma: - IOP is measured by tonometry. - Optic disc or fundus examination (by ophthalmoscope). - Visual field examination (by perimetry: an automated instrument). - Gonioscopy to check angle if it is open or closed (estimates width of angle between iris and cornea). Glaucoma Classification: 1. Congenital Glaucoma 2. Primary Glaucoma a- Open angle b- Closed angle 3. Secondary Glaucoma (due to): a. Inflammation b. Trauma A) Congenital Glaucoma Corneal haze Increased IOP Optic disc cupping Buphthalmos B) Primary Glaucoma 1- Primary open angle glaucoma: - Most common form of glaucoma (affects approximately 95% of individuals) Features: - Painless, common in elderly. 49  - Symptomless. - Trabecular meshwork becomes less efficient at drainage aqueous humor. - Gross field defect (late stages) - Damage to the optic nerve 2. Primary Angle closure glaucoma: - Acute emergency (Must visit ophthalmologist immediately.) - Very painful. - Red eye & eye feels hard. - Reduced blurred vision. - Nausea & vomiting. - Can be triggered by dilating drops. - The drainage angle of trabecular meshwork becomes blocked by the iris. - IOP builds up very fast. Figure ( ): Angle closure glaucoma Treatment Goal of glaucoma: 50  Figure ( ): Goals of glaucoma treatment Approaches of glaucoma therapy: 1- Drug therapy: a. PG analogues: e.g., Latanaprost (Xalatan). b. β-blockers: e.g., Timolol, Betaxalol c. CA inhibitors: e.g., acetazolamide d. Adrenergic agonists: e.g., Apraclonidine e. Cholinergic agonists: e.g., Pilocarpine f. Hyperosmotics: e.g., mannitol 2- Laser trabeculoplasty 3- Surgical treatment First Goal in glaucoma: To lower IOP by: a) Decreasing the production of aqueous humor at the ciliary body. and/ or b) Increasing outflow through the trabecular meshwork pathways. Gilman’s 2006) A) Latanoprost ((Xalatan) 0.005% (once a day) Prostaglandin (PGF2α) analogue. Increases outflow of aqueous humor mediated by effects on ciliary muscles. Once daily adminstration (for 24 hour IOP control) i.e., with persistency & compliance. Leads to a fairly uniform circadian reduction in IOP. Currently , latanoprost is used as first choice therapy for primary open angle glaucoma & ocular Hypertension and other medications are added if necessary. Latanoprost is last choice in: i. Elevated IOP secondary to trauma ii. Inflammatory glaucoma iii. Steroid induced glaucoma N.B. Since Latanoprost decrease IOP by decreasing outflow, adding agent should act by 51  decreasing production as BBs or CAIs Side effects of prostaglandin analogue: Conjunctival hyperemia & Redness Iris pigmentation Foreign body sensation Dark the iris (10%) (green-brown, blue-brown) Eyelashes longer, thicker, heavily pigmented B) Beta adrenergic blockers 1. Non-selective: Timolol (Timoptic ) eye drops twice a day and gel (once a day). 2. Selective: Betaxolol (Betoptic) eye drops (twice a day). Mechanism of action: ββs decrease the production of aqueous humor and have no effect on outflow: – by blocking β1 in ciliary epithlium, → ↓ aqueous humor production. – By blocking β2 receptors in the eye; in cilary process → VC of blood vessels → ↓ production of aquous humor) For non-selective only. Uses: Topically in chronic open angle glaucoma Onset: ≈ 30 minutes & duration ≈ 12 to 24 hours. ADRs of Topical BBs Systemic absorption from topical BBs →: Cardiovascular side effects: bradycardia , heart block & hypotension) Pulmonary side effects: Bronchospasm in susceptible patients. So, patients with bronchial asthma, severe COPD, or bradyarrhythmias are at greater risk of adverse effects. Cardioselective ββ s are less effective than non-selective ββ s but more safe in asthmatics. The second goal in glaucoma is neuroprotection C- ALPHA-2 AGONISTS - Apraclonidine (Iopidine) - Brimonidine (Alphagan) Enhanced α2 selectivity due to double ring structure Both apraclonidine and brimonidine reduce aqueous production and may enhance some outflow. Both appear to bind to pre- and postsynaptic α2 receptors. 52  IOP lowering is the main aim of any anti-glaucoma agent. But, there is something beyond this IOP reduction…. Neuroprotection. Both drugs are neuroprotective and may contribute to visual field preservation in glaucomatous eyes. D- Carbonic Anhydrase Inhibitors Oral: Acetazolamide, methazolamide,... Topical: Dorzolamide (Trusopt TID) or Brinzolamide (Azopt BID). Mode of action: Decrease aqueous humor production by decreasing bicarbonate formation ⇒↓ IOP. Neuroprotective effect may be involved. Side Effects: - Metallic taste - Burning sensation & parasthesia - Acid-base imbalances - Conjonctivitis - Sulfa allergies (usually seen in the antibiotic class of sulfonamides) - Corneal concerns Dorzolamide: Vasoprotection “Vasoprotection” - May be effective in preventing damage resulting from vascular dysfunction of eye - Can lead to improved visual function E) Parasympathomimetics in glaucoma Direct-acting cholinergic agonists: - Pilocarpine (0.5% to 4% solution) or ocular insert (OCUSERT PILO-20) that releases 20 µg of pilocarpine per hour over 7 days. - Pilocarpine was used historically as a standard agent in open-angle glaucoma. Now, used as last choice Indirect-acting cholinergic drugs: - Physostigmine (reversible) & Echothiophate (Irreversible) Mechanism of action: In close angle glaucoma: Miosis Removal of physical obstruction  drainage-- ----- In open angle glaucoma: Contraction of ciliary muscles opening of trabecular meshwork drainage------ ADRs: systemic absorption of cholinergics may produce hypotension, bronchial asthma and bradycardia in susceptible patients. F) Hyperosmotics 53  e.g., Mannitol (Osmitrol), glycerin (Osmoglyn). Mechanism of action: Hyperosmotic means that they increase the movement of fluid through membranes. Hyperosmotics are used in the treatment of acute angle-closure glaucoma. The effects last only 6-8 hours, so they are not for long-term use. Contraindications: Allergy to hyperosmotics, renal disease, severe dehydration, pulmonary edema, severe heart disease, Diabetes. Use: These drugs are given systemically (orally or by injection) to rapidly correct high elevations of intraocular pressure in angle closure glaucoma. The third target: persistency/ compliance - Pharmacologic therapy for glaucoma can be effective only if patients fill their prescriptions (persistency) and take their medications as directed (compliance) * Patients treated with bimatoprost were 31% more likely to discontinue/change therapy compared with Latanoprost * Patients treated with travoprost were 29% more likely to discontinue/change therapy compared with Latanoprost i. The advantage of a once daily dosing regimen in the context of the underlying pharmacological basis that a drug which is enough once a day has a minimal duration of 24 hours. ii. Latanoprost has the advantage of once daily usage. iii. Thus even a mild non-compliance, the occasional “Forgotten drop” will not change the level of IOP very much. Clinical Case 55-year-old male patient, who is a web designer by profession, was referred by their optometrist due to visual field defects. The patient was himself aware of a gradual reduction in his vision over the last few months and therefore went to the optometrist for an eye examination. He reported a positive family history of glaucoma in his maternal grandmother. On examination, this patient’s best corrected distance visual acuity (VA) was 6/60 in the right eye and 6/18 in the left eye. Goldmann tonometry revealed IOPs of 23mmHg in the right eye and 21mmHg in the left eye. The anterior chamber angles as assessed using gonioscopy were open in both eyes. The optic discs were normal in size in both eyes and showed a cup to disc. 1. Which of the following features is unusual for primary open angle glaucoma? a) Optic disc cupping. b) Increased IOP. c) Decrease visual field. 54  d) Positive family history. e) Physical obstruction of the angle of the anterior chamber of the eye. 2. Which of the following is LEAST likely to be a risk factor for retinal vein occlusion? (a) Age of less than 50 years (b) Hypertension (c) Diabetes mellitus (d) Increased IOP 3. Which of the following is an indication for glaucoma surgery? (a) Uncontrolled IOP despite maximal tolerated medical treatment (b) Progressive glaucoma (c) Non-availability and/or unaffordable cost of medications (d) All of the above 4. If this patient asthmatic and hypertensive, you prefer to avoid: a) Timolol b) Pilocarpine c) Mannitol d) All of the above 55  PART-III: DUG SAMPLES Pilocarpine Generic Name Trade Name Mechanism of action Two Similar drugs Three Main Therapeutic Uses Three Main Adverse Effects Three Main Contrindications 56  Neostigmine Generic Name Trade Name Mechanism of action Two Similar drugs Three Main Therapeutic Uses Three Main Adverse Effects Three Main Contrindications 57  Donepezil Generic Name Trade Name Mechanism of action Two Similar drugs Three Main Therapeutic Uses Three Main Adverse Effects Three Main Contrindications 58  Atropine Generic Name Trade Name Mechanism of action Two Similar drugs Three Main Therapeutic Uses Three Main Adverse Effects Three Main Contrindications 59  Ipratropium Generic Name Trade Name Mechanism of action Two Similar drugs Three Main Therapeutic Uses Three Main Adverse Effects Three Main Contrindications 60  Hyoscine (Scopolamine) Generic Name Trade Name Mechanism of action Two Similar drugs Three Main Therapeutic Uses Three Main Adverse Effects Three Main Contrindications 61  Benztropine Generic Name Trade Name Mechanism of action Two Similar drugs Three Main Therapeutic Uses Three Main Adverse Effects Three Main Contrindications 62  Epinephrine Generic Name Trade Name Mechanism of action Two Similar drugs Three Main Therapeutic Uses Three Main Adverse Effects Three Main Contrindications 63  Dobutamine Generic Name Trade Name Mechanism of action Two Similar drugs Three Main Therapeutic Uses Three Main Adverse Effects Three Main Contrindications 64  Salbutamol Generic Name Trade Name Mechanism of action Two Similar drugs Three Main Therapeutic Uses Three Main Adverse Effects Three Main Contrindications 65  Alpha methyldopa Generic Name Trade Name Mechanism of action Two Similar drugs Three Main Therapeutic Uses Three Main Adverse Effects Three Main Contrindications 66  Prazosin Generic Name Trade Name Mechanism of action Two Similar drugs Three Main Therapeutic Uses Three Main Adverse Effects Three Main Contrindications 67  Tamsulosin Generic Name Trade Name Mechanism of action Two Similar drugs Three Main Therapeutic Uses Three Main Adverse Effects Three Main Contrindications 68  Propranolol Generic Name Trade Name Mechanism of action Two Similar drugs Three Main Therapeutic Uses Three Main Adverse Effects Three Main Contrindications 69 Generic Name Trade Name Mechanism of action Two Similar drugs Three Main sesTherapeutic U Three Main Adverse Effects Three Main Contrindications 70 Generic Name Trade Name Mechanism of action Two Similar drugs Three Main sesTherapeutic U Three Main Adverse Effects Three Main Contrindications 71