Lab Manual for Practical Pharmacology.pdf

Ministry of Higher Education ‫وزارة التعليم العالي‬ & Scientific Research ‫والبحث العلمي‬ Al-Zahrawi University College ‫كلية الزهراوي الجامعة‬ Department of Pharmacy ‫قسم الص...

Ministry of Higher Education ‫وزارة التعليم العالي‬ & Scientific Research ‫والبحث العلمي‬ Al-Zahrawi University College ‫كلية الزهراوي الجامعة‬ Department of Pharmacy ‫قسم الصيدلة‬ Lab Manual for Practical Pharmacology 4th class By Asst. lec. Mohammed Shaheed Asst. lec. Muntadher Zyara Lab 1 Routes of drug administration Definition: Routes of drug administration defined as the mean by which drugs can be delivered into the body. Factors to be considered when choosing certain route: 1. Site of drug action: e.g., treatment of certain GIT diseases necessitates giving the drug orally. 2. Drug nature: intravenous fluids should be isotonic. 1  3. Onset of action: treatment of emergency conditions necessitates the use of intravenous route. 4. Duration of action: drugs intended for longer duration of action are given by a route when absorption is slow. 5. Patient status: oral route cannot be used when the patient is unconscious, or has difficulty in swallowing, or has repeated vomiting. 6. Desire of the patient: patient compliance. 1. Oral route: By placing mouth gag (needle gavage) into the mouth of rat or rabbit. To make sure that the tube is in the esophagus and not in the trachea, dip the end of the tube into a beaker containing water (bubbling indicates wrong position). By using medical syringe, push 2.5 ml of normal saline (N.S) into the stomach through the rubber tube. 2 Advantages: Simple, convenient, and acceptable. Oral drugs can be given in different dosage forms. Safe route: since in overdose, it can be managed easily. The drug can be placed at the site of action (e.g., anthelminthics). Disadvantages: Some drugs are destroyed in the gut (e.g., some penicillins, insulin, oxytocin) Tablets taken with too small a quantity of liquid and in supine position can lodge in the esophagus with delayed absorption and may even cause ulceration (e.g., doxycycline). Some drugs may cause gastric irritation. Absorption may be affected by food or by other drugs which inhibit gut motility e.g., antimuscarinic and opioids. 3  First pass metabolism (by intestinal wall and by the liver) limits the efficacy of some drugs when taken orally. 2. Intravenous (IV) route: Advantages: Large volumes can be given via this route. Unconscious patient. Rapid onset of action. Suitable for continuous infusion (for rapidly destroyed drugs) Suitable for too irritant drugs (anticancer drugs). No first pass metabolism. Disadvantages: IV fluid should be aqueous, and (isotonic) Infection and local venous thrombosis. The injected drug cannot be recalled simply in case of toxicity. 4 3. Intramuscular (IM) route: Common route, and less affected by peripheral circulatory failure. Preparation is about 2 ml, and isotonicity is not essential except for the comfort of the patient. Drug can be aqueous or specialized depot preparations. Sites of injection: gluteal region in the upper lateral quarter. Advantages: More rapid than subcutaneous route as the absorption is more rapid. Unconscious patient. Disadvantages: Not acceptable for self-administration May be painful. 4. Subcutaneous (SC) route: The solution to be injected should be aqueous and isotonicity is not essential except for the comfort of the patient. Common route for the administration of insulin. Poor absorption in case of peripheral circulatory failure. Advantage: reliable and acceptable for self-administration. 5 Disadvantage: skin reaction which can be avoided by changing site of administration. 5. Intraperitoneal (I.P) route: Relatively large volume of non-irritant drugs. Absorption is faster in the peritoneal cavity than IM or SC. Site of injection In mice and rats: in the lower half of abdomen. In rabbits: in the lower left quadrant of abdomen to avoid injuring the liver. Hold the animal and insert needle at angle of 45 degrees. 6. Inhalational route: The drug is taken through the inspired air (gas, or aerosol). Rapid absorption (wide surface area) with rapid effect almost as rapid as IV route. Particularly effective for respiratory disorders because the drug is delivered directly at the site of action and systemic S.E. are minimized. Put a piece of cotton soaked with ether in a closed glass container, place rat or rabbit inside the jar and observe it. Whenever there is a change in behavior, remove the animal. Advantages Self-administration. Close control of the dose. 6  Rapid onset due to wide area of absorption. Disadvantages: Need special apparatus Should not be irritant if the patient is conscious. Study exam 1. What is the ideal route for the administration of most drugs used in emergency life support situations? why? 2. Why the absorption of drugs of the intramuscular route is faster than the subcutaneous route? 7 Lab: 2 Effect of Parasympathomimetic Drugs on Glandular Secretions in Rats Autonomic Nervous System The autonomic nervous system conveys all the outputs from the CNS to the rest of the body, except for the motor innervation of skeletal muscle Compose of two neurons, the pre-ganglionic neuron with cell body in the CNS and the post-ganglionic neuron with cell body in the autonomic ganglia The main processes that it regulates are: ✓ Control circulation, respiration, digestion, and body temperature ✓ Contraction and relaxation of vascular and visceral smooth muscle ✓ All exocrine and certain endocrine secretions ✓ The heartbeat ✓ Energy metabolism Three main parts sympathetic parasympathetic The enteric nervous system The Parasympathomimetic nervous system innervates a large number of organs. The neurotransmitter acetylcholine (Ach) mediates the transmission of impulses from the preganglionic neurons to postganglionic neurons as well as the transmission of impulses from postganglionic nerve terminals to the effector organs. 8  Preganglionic neurons of the sympathetic and parasympathetic divisions and postganglionic neurons of the parasympathetic nervous system utilize acetylcholine (ACh). Postganglionic neurons of the sympathetic nervous system use norepinephrine and epinephrine. Sympathetic ganglia are located in the paravertebral chain, thus they have short preganglionic fibers and long postganglionic fiber The axons of the parasympathetic preganglionic neurons are quite long compared to those of the sympathetic system with short postganglionic neurons The action of Ach at the effector organ can be mimicked by drugs like Carbachol, methacholine, or muscarine. The sites at which Ach and the Parasympathomimetic act are called Muscarinic receptors (M receptors) and they are sensitive to block by atropine. Ach is the only neurotransmitter of this system that can act on two types of receptors: Nicotinic and Muscarinic Muscarinic Receptors ✓ G. protein coupled receptors ✓ Five types of these receptors (M1-M5): M1 receptors (neural): C.N.S., gastric parietal cells, they mediate an excitatory effect result an increase of acid secretion M2 receptors (cardiac);present in the heart, they exert an inhibitory effects causing bradycardia M3 receptors (glandular);present in the smooth muscle of G.I.T., bronchial, bladder and exocrine glands; like sweat gland, salivary and 9  lacrimal gland. They mediate excitatory effect which increases the glandular secretion and contraction of visceral smooth muscle M4 and M5 receptors; present in C.N.S. but their functions are not fully identified parasympathomimetic Drugs Direct acting drugs; Choline esters like: METHACHOLINE CARBACHOL BETHACHOLINE Indirect acting drugs; Cholinesterase inhibitors o Reversible PHYSOSTIGMINE, NEOSTIGMINE, PYRIDOSTIGMINE o Irreversible: ORGANO PHOSPHATES Antimuscarinic agents Antimuscarinic agents Block muscarinic receptors cause an inhibition of all muscarinic function 10  Have little or no action at skeletal N.M.J. or autonomic ganglia, they do not block Nicotinic receptors. Reverse excessive cholinergic effects (reverse DUMBBELS)? ❑ Atropine (Hyoscyamine) Orally absorbed, cross BBB Competitive antagonist to Ach at all muscarinic receptors Scopolamine: anti-motion sickness Pirenzepine: block M.R. of the gastric parietal glands; hence it is preferred in the treatment of gastric ulcer Ipratropium: useful in the treatment of asthma to decrease secretion & dilate obstructed air ways Effect of Parasympathomimetic Drugs on Glandular Secretions in Rats Ach and parasympathomimetic stimulates the secretion of different glands in the body, like mucous, sweat, salivary and tear glands as well as secretory activity of the stomach, intestine and pancreas. In the rat, there is a special horseshoe-shaped gland is located within the bony orbit called Harderian gland. This gland Involves muscarinic receptors. 11 The Harderian gland The Harderian gland (or Harder's lacrimal gland) is an exocrine gland, horseshoe-shaped located within the bony orbit Secretions from this gland include the reddish pigment porphyrin Harderian secretions coat the eye, and drain down into the nose through the nasolacrimal duct. A rat may smear the secretions around his nose Involves muscarinic receptors ❑ Hypersecretion in stressed rats is often referred to as "red tears" (chromodacryorrhea) ❑ Rats overproduce porphyrin when they are: Stressed, ill, or poorly fed Water deprivation Joint pain , morphine withdrawal 12  Acetylcholine injections After injection with acetylcholine, for example, profuse amounts of porphyrin were secreted almost immediately and overflowed the eye to stain the eyelids within minutes Cholinergic agonists such as neostigmine, carbachol, and pilocarpine caused chromodacryorrhea Atropine blocks chromodacryorrhea induced by systemic administration of direct-acting cholinergic agonists !! Thus, chromodacryorrhea appears to be a muscarinic receptor related event Neostigmine Neostigmine is a medication used to treat myasthenia gravis and urinary retention without the presence of a blockage. It is also used in anesthesia to end the effects of non-depolarising neuromuscular blocking medication. It inhibits the hydrolysis of acetylcholine by competing with acetylcholine for attachment to acetylcholinesterase at sites of cholinergic transmission. It enhances cholinergic action by facilitating the transmission 13 Experimental protocol Procedure: 1. Inject of 0.12 mg/kg of neostigmine I.P into a rat. 2. Examine the eyes for tears by wiping the eyelids with cotton to detect the bloody tears. Note salivation and nasal secretion…..etc. 3. Inject another rat with 0.5 mg/kg of atropine I.P, wait about 15-25 minutes, and inject the rat with o.12 mg/kg neostigmine I.P. 4. Examine for bloody tears, salivation and nasal secretion….etc Aim of the experiment 1. This exp. was designed to show the stimulatory effect of cholinergic drug on glandular secretion 2. To prove that these glands are parasympathetically innervated and contain a muscarinic type of receptors Study exam 1. What is DUMBBELS? 2. What is Neostigmine? And on which receptors does it work? 3. What is Atropine? And on which receptor does it work? 14 Lab 3 Drug antagonist (Histamine and its antagonists) Aim: To show the effect of Histamine and its antagonists on the human skin. ✓ Agonist, Partial Agonist, Antagonist, Inverse Agonist: Agonist (full agonist): drugs or endogenous substances (neurotransmitters, hormones) that binds to and activates a receptor and produces a biologic effect. For example, morphine act as full agonist at μ opioid receptor. partial agonist: an agonist that is not capable to produce a maximal response. For example, Buprenorphine is a partial agonist at the μ receptor. Antagonists: is a molecule bind with receptor but produce no activation of the receptor. For example, Atropine blocks the action of Acetylcholine (Ach) in mAChR. Inverse agonist: is a molecule that binds with a receptor and decreases the baseline activity of the receptor, which in turn produces a reverse effect that of binding of the agonist. For example, nalmefene was identified as inverse agonists at opioid receptor. 15 Histamine: is a chemical messenger that mediates a wide range of cellular responses, including allergic and inflammatory reactions, gastric acid secretion, sleep and wake cycle, cognitive ability and food intake. Location: All tissues. High amounts in lung, skin and GIT (where the inside meets the outside) High concentration in mast cells and basophiles It is a component of venoms and found in secretions from insect stings. Release: Stimuli: 1. Destruction of cells as a result of cold, toxins, bee sting venoms, or trauma. 2. Allergic and anaphylactic reactions (combination of antigen with cell-fixed immunoglobulin (Ig)E antibodies). Mechanism of action: Histamine acts by binding to one or more of its receptors: 1. H1 receptors: mediate edema and vascular effects of histamine. Antagonists: 16  H1 antagonists (e.g., diphenhydramine) 2. H2 receptors: mediates the effects on gastric secretion. Antagonists (e.g. cimetidine) 3. H3 receptors. 4. H4 receptors. Effects of histamine: Clinically important effects of histamine are those on smooth muscles, blood vessels, skin, and secretary glands. IV injection (even small amount e.g., 0.1 mg) will produce the followings; systemic vasodilation with rapid fall in blood pressure, increased heart rate, flushing of face, headache, stimulate gastric acid secretion, these changes last for few minutes. Intradermal injection (even of a relatively high amount e.g., 10 mg) will produce: Triple response of Lewis: which consist of the followings: 1. Flush: localized erythema due to dilation of capillaries. 2. Wheal: localized edema due to increased permeability of capillaries and post capillary venules. 3. Flare: erythema caused by arterial dilation, (it is due to axon reflex). Histamine antagonists: The effects of histamine can be opposed in 3 ways: 17 1. By using a drug with opposite effects e.g., histamine constricts bronchi, causes vasodilation and increase capillary permeability. Adrenaline opposes these effects by a mechanism unrelated to histamine (α and β receptors). This is a physiological antagonism. 2. H1, H2 receptor antagonists (competitive) (preventing histamine from reaching its sites of action) 3. by preventing the release of histamine from cells in which it is stored: adrenal corticosteroids and sodium cromoglycate (membrane stabilizing action) Role of histamine in allergy and anaphylaxis: The symptoms resulting from IV injection of histamine are similar to those associated with anaphylactic shock and allergic reactions. Anaphylactic shock occurs with penicillins and other drugs. ✓ Signs and symptoms of anaphylactic shock: severe hypotension, bronchospasm, edema, (including laryngeal edema), and sometimes death due to loss of fluid from the intravascular compartment. Drugs used in treatment of anaphylactic shock: 4. Adrenaline. 5. Glucagon: in patient that are taking B-blocker appear to refractory to adrenaline. 6. antihistamines: (e.g., diphenhydramine). 18  7. Corticosteroids (e.g., Hydrocortisone, methylprednisolone) 8. IV fluid infusion. Procedure of the experiment: 1. Cleanse the forearm with an alcohol-soaked cotton wool. 2. the following drugs are placed on skin and a prick is made through the drop of the drug: a) One drop of histamine (1: 1000) solution. b) One drop of histamine and one drop of adrenaline (1:1000) solution. c) Two drops of diphenhydramine and one drop of histamine d) Observe the response. Note: 1- To obtain better result, it is preferable to do the above procedure on persons with fair skin. 2- This experiment is not to be done on atopic individuals. Exp. Needs: N.S, anti-histamine, adrenaline, histamine, Drops, lancet, cotton, alcohol. Study exam: 1. In the histamine experiment, what is the effect of adrenaline drops on scratched skin? Explain your answer. 2. What does physiological antagonism mean? Explain your answer with an example 19 Lab.4 Acute toxicity Acute toxicity: - describes the adverse effects of a substance that result either from a single exposure or from multiple exposures in a short space of time (usually less than 24 hours). To be described as acute toxicity, the adverse effects should occur within 14 days of the administration of the substance. Acute toxicity is distinguished from chronic toxicity, which describes the adverse health effects from repeated exposures, often at lower levels, to a substance over a longer time period (months or years). It is widely considered unethical to use humans as test subjects for acute (or chronic) toxicity research. However, some information can be gained from investigating accidental human exposures (e.g., factory accidents). Otherwise, most acute toxicity data comes from animal testing or, more recently, in vitro testing methods and inference from data on similar substances. Benzodiazepines The commercial use of benzodiazepines began with the introduction of chlordiazepoxide for anxiety in 1961 and shortly thereafter of diazepam for seizures in 1963. Benzodiazepines are used principally as sedatives. Temazepam and triazolam are exceptions; they are used as hypnotics to produce sleep. Clonazepam is the only benzodiazepine approved for use as a chronic anticonvulsant agent. 20 The benzodiazepines are organic bases with a benzene structure and a 7-member diazepine moiety. Similar to barbiturates, various side chains at R1, R2, R2’, R3, R4, R5, and R7 influence potency, duration of action, metabolites, and rate of elimination. Toxicokinetic: Benzodiazepines tend to be highly protein bound and lipophilic. They passively diffuse into the CNS, their main site of action. Because of their lipophilic nature, benzodiazepines are extensively metabolized via oxidation and conjugation in the liver prior to their renal elimination. Benzodiazepine receptors: Benzodiazepines associated with GABA receptors at specific areas in the CNS. Two structurally different [central] benzodiazepine receptors are found in the brain: type I and type II. Type I receptors tends to be located throughout the brain and contain the GABAA subunit. They are hypothesized to affect anxiety, sleep, and amnesia. Type II receptors are concentrated predominantly in the hippocampus, striatum, and the spinal cord. They are hypothesized to affect muscle relaxation and dependence. 21 Peripheral benzodiazepine receptors: Benzodiazepines are also active at certain types of benzodiazepine receptors that are not associated with the GABA receptor. These receptors differ structurally, pharmacologically, and physiologically from GABA-associated benzodiazepine receptors. The function and structure of these receptors are not well defined Several types of endogenous benzodiazepine like substances, endozepines, are proposed to bind to these receptors. Peripheral benzodiazepine receptors may play significant role in modulating pathologic conditions such as hepatic encephalopathy, anxiety disorders, and abnormal immune function. Tolerance and Dependence: Tolerance to the sedative effects of the benzodiazepines occurs more rapidly than does tolerance to the antianxiety effects. Abrupt discontinuation following long-term use of benzodiazepines may precipitate benzodiazepine withdrawal, which is characterized by autonomic instability, changes in perception, paresthesias, headaches, tremors, and seizures. Alprazolam and lorazepam are associated with more severe withdrawal symptoms and more frequent recurrent symptoms compared with chlordiazepoxide and diazepam, drugs that may protect the user because of the effects of their active metabolites. 22 Overdose: A unique property of the benzodiazepines is their relative safety even after substantial ingestion, which probably results from their GABA receptor properties. Unlike many other sedative-hypnotics, benzodiazepines do not open GABA channels independently at high concentrations. Benzodiazepines are not known to cause any specific systemic injury, and their long-term use is not associated with specific organ toxicity. Deaths resulting from benzodiazepine ingestions alone are extremely rare. Most often deaths are secondary to a combination of alcohol or other sedative hypnotics. Supportive care is the mainstay of treatment. Flumazenil Flumazenil is a water-soluble benzodiazepine analog with a molecular weight of 303 Daltons. It is a competitive antagonist at the benzodiazepine receptor, with very weak agonist properties in animal models and in humans. The benzodiazepine receptor modulates the effect of GABA on the GABAA receptor by increasing the frequency of Cl- channel opening, leading to hyperpolarization. Agonists such as diazepam stimulate the benzodiazepine receptor to produce anxiolytic, anticonvulsant, sedative, amnestic, and muscle- relaxant effects at low doses and hypnosis at high doses. Inverse agonists bind the benzodiazepine receptor and result in the opposite effects of anxiety, agitation, and seizures. Antagonists, such as flumazenil, competitively occupy the benzodiazepine receptor without causing any functional change and without allowing an agonist or inverse agonist access to the receptor. 23 Investigations reveal that 1.5 mg flumazenil leads to an initial receptor occupancy of 55%, whereas 15 mg causes almost total blockade of benzodiazepine receptor sites. Physicochemical and Pharmacologic Properties of Flumazenil 1. pKA Weak base 2. Volume of distribution 1.06 L/kg 3. Distribution half-life 5 minutes 4. Metabolism Hepatic: three inactive metabolites 5. High clearance 6. Elimination First order 7. Protein binding 54-64% 8. Elimination half-life 53 minutes 9. Onset of action 2 minutes 10.Duration of action Dependent on dose and elimination of benzodiazepine, time interval, dose of flumazenil, and hepatic function The reversal effects of Flumazenil: Volunteer studies demonstrate the ability of flumazenil to reverse the effect of benzodiazepines. Reversal is dose dependent and begins within minutes. Peak effects occur within 6-10 minutes. Most individuals achieve complete reversal of benzodiazepine effect with a total IV dose of 1 mg. 24 Use in Benzodiazepine Overdose: Flumazenil has no role in cases of unknown overdose because seizures and dysrhythmias may occur when the effects of a benzodiazepine are reversed in a mixed overdose. Flumazenil appears safe and effective for reversal of sedation and partial reversal of amnesia and cognitive impairment. Flumazenil does not reliably reverse the respiratory depression induced by intravenous benzodiazepines but does reverse central nervous system (CNS) depression. Flumazenil also has the potential to induce benzodiazepine withdrawal symptoms, including seizures in patients who are benzodiazepine dependent. Experimental work: Procedure: - IP injection of 0.2 mg Diazepam in mice 1 IP injection of 0.4 mg Diazepam in mice 2 Observe the effects for 6-10 min. IP injection of 20 µg Flumazenil in mice 1 IP injection of 40 µg Flumazenil in mice 2 Observe the effects for 3-5 min. Discussion: What are the expected effects in mice 1?? 25 What are the expected effects in mice 2?? Contraindications to Flumazenil Use Prior seizure history or current treatment of seizures History of ingestion of a xenobiotic capable of provoking seizures or cardiac dysrhythmias Long-term use of benzodiazepine 26  Lab. 5 Evaluation of antipsychotic drugs haloperidol (catalepsy) Antipsychotics are a type of psychiatric medication which are available on prescription to treat psychosis. They are licensed to treat certain types of mental health problem whose symptoms include psychotic experiences. This includes: schizophrenia schizoaffective disorder some forms of bipolar disorder severe depression the psychotic symptoms of a personality disorder. Some antipsychotics are also licensed to treat other health problems, including: physical problems, such as persistent hiccups, problems with balance and nausea (feeling sick) agitation and psychotic experiences in dementia. This is only recommended if you pose a risk to yourself or others, or if you are severely distressed. Antipsychotics can be prescribed to be taken in various different ways. Most commonly you will take them by swallowing them, in tablet or liquid form. But some of them can also be prescribed as a depot injection. This is a slow-release, slow-acting form of the medication, given as an injection every few weeks. 27  If you are given antipsychotics in hospital, doctors may use a type of antipsychotic that you can inhale, called loxapine adasuve. But this is not available for general prescription. Catalepsy a medical condition characterized by a trance or seizure with a loss of sensation and consciousness accompanied by rigidity of the body. ✓ Haloperidol is a drug used to induce model of catalepsy. 28 Lab:6 Barbiturates Thiopental and Dose Calculation Calculate Your Dose The dose is the amount of drug taken at any one time. Weight of drug (e.g. 250 mg) Volume of drug solution (e.g. 10 mL, 2 drops) The number of dosage forms (e.g. 1 capsule, 1 suppository) Other quantity (e.g. 2 puffs) The dosage regimen is the frequency at which the drug doses are given. Ex. 2.5 mL twice a day, one tablet three times a day… Accurate dosing is critical for the proper utilization of all pharmaceuticals. ✓ First you need to know what volume you want to inject into the animal with each treatment being administered, then you need to know how much drug should be in that given volume To calculate the correct dose of drug you need to know: I. The concentration of the drug II. The weight of the animal III. The recommended dose rate of the drug for each specific animal model 29 Concentration of the drug: mg/ml: Manufacturers usually provide concentrations of their product in milligrams (mg) of drug per (ml) of solvent % : grams per 100 ml. ex: 10% solution of xylazine is 100 mg/ml IU/ml: International Units per ml of, like some of the fat soluble vitamins Powders: The mg of active drug in the vial. For example, Telazol (tiletamine and zolazepam) comes in powdered form with 500mg per vial: If you add 5ml of sterile water for injection to the vial thus providing 5ml of 100mg/ml drug If you add 2.5ml of sterile water for injection, will make 2.5ml of a 200mg/ml solution. Weight of the animal: It is always best to use a scale and get an accurate weight. If you cannot weigh the animal prior to injection, you need to be experienced in estimating the weight. Dose rate of the drug: Always look up the drug dose for the species you are working with - it often varies. For most applications the following formula is applicable: (C1)(V1) = (C2)(V2) Ex. You have 20 ml of a 10 mg/ml solution and you want to make 15 ml of a 2.5 mg/ml solution. Set up the math as follows: C1 = 10 mg/ml C2 = 2.5 mg/ml V1 = unknown V2 = 15 ml (10 mg/ml) (V1) = (2.5 mg/ml) (15 ml) 30  V1 = 3.75 ml So you dilute 3.75 ml of C1 to a final volume of 15 ml therefore you need to add: 15 - 3.75 =11.25 ml of diluent. Let's say you want to treat 15 rats with 75 mg/kg of a compound at the rate of 0.1 ml/20 gm of body weight and you want to prepare enough drug to dose 4 days, weight of each rat is 100 gm. Number of animals= 15 Dose to each animal= 75 mg/kg OR 7.5 mg/100gm Volume injected to each rat= 0.1ml/20 gm OR o.5 ml/100 gm Thus, each 7.5 mg compound should be dissolved in 0.5 ml vehicle (for one rat) (7.5mg/0.5 ml) x 15 = 112.5 mg/7.5ml (for 15 rats in each day) (112.5mg/7.5ml) x 4 = 450 mg/ 30 ml (to 15 rats for 4 days) SO, we weight 450 mg compound and dissolve it in 30 ml vehicle, and inject each rat with 0.5 ml of the solution for 4 days. Barbiturates ❖ Class of drugs that act as central nervous system depressants, and can therefore produce a wide spectrum of effects, from mild sedation to total anesthesia. ❖ Long acting barbiturates, ex. Phenobarbital. which has 6-8 hrs (duration of action), 4-5 days elimination half-life. ❖ Short acting barbiturates, ex. Butobarbital and Pentobarbital. 2-4 hrs.(duration of action) 31  ❖ Ultra short acting barbiturates, ex. Thiopental. (Duration of action 20-25 min, h1/2=8-10 hrs.) ❖ They are also effective as anxiolytics, hypnotics, and anticonvulsants. ❖ Barbiturates also have analgesic effects, however these effects are somewhat weak, preventing barbiturates from being used in surgery in the absence of other analgesics. ❖ They have addiction potential, both physical and psychological CNS depressant effects of barbiturates: ▪ Used in anesthesia, insomnia and the treatment of epilepsy and seizure disorders. ▪ Respiratory Effects are dose-dependent: i. At hypnotic doses, little effects. ii. At high doses, depress respiratory center iii. Death due to respiratory failure Barbiturates have now largely been replaced by benzodiazepines in routine medical practice, mainly because: 32  1. benzodiazepines are significantly less dangerous in overdose. barbiturate shortened the time taken to sleep and lengthened the delay to rapid eye movement (R.E.M.) sleep) Quick tolerance High potential for dependence and abuse (addiction). Potent inducer for liver metabolizing enzymes and drug interactions. Mechanism of action GABA Modulators: Enhance binding of inhibitory NT GABA with its receptors GABA-A binding site. they cause allosteric modulation of GABA-action on GABA-A receptor, prolonged the duration of the GABA-gated Cl-channel opening (influx of Cl) hyperpolarization CNS depression. At higher concentration: Barbiturate directly increase Cl- conductance (GABA mimetic action; contrast BZDs which have only GABA facilitatory action). Block the AMPA receptor (a subtype of glutamate receptor), Leading to decrease the activity of excitatory glutamate neurotransmitter and depress the neuronal depolarization effect. They inhibit the Ca+2 -dependent release of neurotransmitters. 33  The action of barbiturates is non-selective i.e. increase dose of barbiturates generalized CNS inhibition. Pharmacokinetics: High lipid solubility: Cross blood brain barrier, rapid onset. Redistribution to other tissues outside the CNS :Short duration of action. Liver: All metabolized into inactive metabolites except (Phenobarbital excreted unchanged) Renal excretion. Alkalization of urine in case of toxicity. Adverse effects: Drowsiness, disorientation, respiratory depression. Tolerance: Decreased responsiveness to the drug upon repeated administration. Dependence: both psychological and physiological. Withdrawal is much more sever than that associated with opiates and can result in death. Peripherally: hypotension due to myocardial depression and depression of VMC Potent inducer for liver metabolizing enzymes. Chronic use of barbiturates will cause upregulation, or induction, of the microsomal enzymes (CYPs) ,increasing the metabolism of endogenous substrates and other drugs metabolized by these enzymes. This can lead to patients requiring larger dosages of medication to achieve therapeutic effect and/or increased clearance This enzyme induction also causes barbiturate tolerance due to increased barbiturate metabolism. 34 Experimental protocol Thiopental 1 gm vial Thiopental , 40mg/kg I.P in mice. 6 mice receive I.P Measured Parameters 1. General Activity 2. Characteristics of Breathing 3. Onset of Sleep (mins) 4. Duration of Sleep (mins) ✓ Barbiturates are hypnotic drugs: Onset of action is the time required to loss the righting reflex. Duration of action in mice can be measured by the ‘sleeping time’ (i.e. the time from the loss of righting reflex to recovery of reflex). Experimental protocol The loss of righting reflex (LORR) assay is used to evaluate sedative/hypnotic effects. Righting reflex: the ability to assume an optimal position when there has been a departure from it The onset time of sleep was noted for all animals. After induction of sleep, mice were placed in the inverted position and when sedation was over, the mice came to normal posture and time was noted Record: LORR was recorded as the time at which the animal was unable to turn itself (onset of action) 35  The time to regain the righting reflex (duration of action, DOA) Report: Name & aim of experiment: You need to write the name of experiment and the aim of the work, for example: We aimed to investigate and evaluate the sedative/hypnotic effects. Thiopental administered IP in Mice. Methods: Mention the animal used, the drug injected with the doses. Describe the work. For example: six mice were injected with thiopental 40 mg/kg IP. The LORR and duration of sleep were recorded………etc. Results: onset of action/duration of action Discussion: mention and discuss your results, for example: From the results obtained, we noted that onset of action was faster in IP than SC route. This is due to…..etc. 36 Lab. 7 Effects of Narcotic Analgesic Drugs Opioids analgesics History of Opioids Opium is extracted from poppy seeds used for thousands of years to produce: ✓ Euphoria, Analgesia, Sedation, Relief from diarrhea, Cough suppression Terminology: “opium” is a Greek word meaning “juice,” or the exudate from the poppy “opiate” is a drug extracted from the exudate of the poppy “opioid” is a natural or synthetic drug that binds to opioid receptors producing agonist effects Natural opioids occur in 2 places: 1. In the juice of the opium poppy (morphine and codeine). 2. As endogenous endorphins. All other opioids are prepared either from morphine (Heroin) or synthesized from precursor compounds (Fentanyl) Agonists: Morphine: prototype Heroin: Diacetylmorphine Fentanyl: potent analgesic Codeine: cough suppression Methadone: synthetic 37  Pethidine: synthetic, like morphine Diphenoxylate:(lomotil) Tramadol: synthetic Antagonists: Naloxone, Naltrexone Opioid Receptors: 1. Mu (µ) ✓ µ1: responsible for analgesia ✓ µ2: responsible for respiratory depression, spinal analgesia, physical dependence, and euphoria. 2. Kappa receptors: responsible for modest analgesia, Dysphoria 3. Delta receptors: unclear action. Pharmacological Effects: Sedation (Drowsiness, lethargy, Apathy) Depression of respiration -Main cause of death from opioid overdose -Combination of opioids and alcohol is especially dangerous Cough suppression: Suppress the “cough center” in the brain Pupillary constriction: Pin point pupil is characteristic of opioids toxicity. 38 Clinical Uses: relief pain (traumatic, visceral, biliary, renal,) suppress cough (codeine) stop diarrhea pulmonary edema (acute left vent. Failure) to comfort dying patient (CA) Tolerance: Occurs rapidly after repeated doses of Morphine Cross tolerance among related drugs like Pethidine Decrease in intensity and shortening of duration of sedation and euphoria Tolerance dose can reach 50-60 times the normal dose (10mg) that can kill the patient if stop taking the drug. Dependence: Physiological dependence occurs when the drug is necessary for normal physiological functioning, this is demonstrated by the withdrawal reactions that are usually the opposite of the physiological effects produced by the drug. Method 1: 1- Inject 6 mg/kg morphine (I.P) to one rat 2- Check the following parameters before and after injection: ✓ Righting reflex ✓ Pain reflex ✓ Gait 39  ✓ Erection of tail (or what is called Straub's phenomenon, which occurs due to stimulation of the spinal cord by the narcotics). Method 2: 1. Measure the weight of two mice to determine the dose of drugs. 2. The 1st mouse received normal saline and consider as a control, the 2nd mouse received 6 mg/kg morphine (I.P) 3. After 10 minutes of drug administration, the animals were placed on Eddy's hot plate kept at a temperature of 55 Co with a cut off period of 15 seconds, was observed to avoid damage to paw. 4. Reaction time was recorded when animals licked their fore or hind paws, or jumped prior to 0, 30, 60 and 120 minutes after administration of drugs. 40 Lab:8 Evaluation of antianxiety drugs clonidine plus maze Overview Experiencing occasional anxiety is a normal part of life. However, people with anxiety disorders frequently have intense, excessive and persistent worry and fear about everyday situations. Often, anxiety disorders involve repeated episodes of sudden feelings of intense anxiety and fear or terror that reach a peak within minutes (panic attacks). These feelings of anxiety and panic interfere with daily activities, are difficult to control, are out of proportion to the actual danger and can last a long time. You may avoid places or situations to prevent these feelings. Symptoms may start during childhood or the teen years and continue into adulthood. Examples of anxiety disorders include generalized anxiety disorder, social anxiety disorder (social phobia), specific phobias and separation anxiety disorder. You can have more than one anxiety disorder. Sometimes anxiety results from a medical condition that needs treatment. Types of anxiety medication Several types of medication can treat the symptoms of anxiety. According to the Anxiety and Depression Association of America (ADAA), the four major classes of drugs for anxiety disorders are: 1. Selective serotonin reuptake inhibitors 41 Although selective serotonin reuptake inhibitors (SSRIs) are a type of antidepressant, doctors can prescribe them to people with anxiety and obsessive- compulsive disorder (OCD). SSRIs work by stopping nerve cells in the brain from reabsorbing serotonin, which is a chemical that plays a vital role in mood regulation. Examples of SSRIs for anxiety include: citalopram escitalopram fluoxetine fluvoxamine paroxetine sertraline 2. Serotonin-norepinephrine reuptake inhibitors Serotonin-norepinephrine reuptake inhibitors (SNRIs) are another class of antidepressant that treats depression and anxiety. Doctors may also prescribe them to treat some chronic pain conditions. The ADAA notes that medical professionals also consider SNRIs to be the first- line treatment for anxiety. However, they are not as effective in treating OCD. These medications work by reducing the brain’s reabsorption of the chemicals serotonin and norepinephrine. 42 Examples of SNRIs for anxiety are: duloxetine venlafaxine As with SSRIs, SNRIs can take several weeks to have an effect. Learn more about SNRI drugs here. 3. Tricyclic antidepressants Tricyclic antidepressants (TCAs) are an older class of antidepressant drug. Although they may be effective for the treatment of depression and anxiety, doctors often prescribe SSRIs instead as they cause fewerTrusted Source side effects. However, TCAs may be useful for some people, especially if other medications do not provide relief. These medications work by blocking the reabsorption of serotonin and norepinephrine. This increases the levels of these neurotransmitters in the brain. Examples of TCAs for anxiety include: amitriptyline imipramine nortriptyline 4. Benzodiazepines Benzodiazepines are a type of sedative drug that reduces the physical symptoms of 43 anxiety, such as tense muscles. These drugs also encourage relaxation, and their effects take place quickly. Benzodiazepines include: alprazolam chlordiazepoxide diazepam lorazepam Although they are highly effective for short-term issues, doctors rarely prescribe benzodiazepines because they become less effective over time and can be addictive. Some people may take benzodiazepines to manage short-term anxiety. For example, people with a fear of flying may take them before a flight. Other medications for anxiety Many other medicines may help treat anxiety, although doctors usually only prescribe them if SSRIs or similar drugs do not work. Other medications for anxiety include: Beta-blockers Beta-blockers are a common medication for people with high blood pressure and heart conditions. However, doctors may prescribe them off-label for anxiety in certain situations. Beta-blockers reduce the effects of norepinephrine, meaning that they can relieve 44 some of the physical symptoms of anxiety. Examples of beta-blockers include atenolol (Tenormin) and propranolol (Inderal). Buspirone This anti-anxiety medication may treat short- or long-term anxiety symptoms. Buspirone (BuSpar) works much more slowly than benzodiazepines and may not treat all types of anxiety disorder, but it causes fewer side effects and has a lower risk of dependency. Monoamine oxidase inhibitors Monoamine oxidase inhibitors (MAOIs) are one of the earliest types of antidepressant. Doctors may prescribe them off-label to treat the symptoms of panic disorder and social phobia. Types of MAOI include: isocarboxazid phenelzine selegiline tranylcypromine 45 Lab. 9 Drugs acting on the eye The main compartments of the human eye (as shown in figure 1) are cornea, iris, lens, ciliary body and vitreous humour. Figure 1: The composition of the human eye Iris: That involves: ✓ Circular muscle (Muscarinic receptors). ✓ Radial muscle (Alpha-receptors). Miosis: is due to either contraction of circular muscle or relaxation of radial muscle. Mydriasis: is due to either contraction of radial muscle or relaxation of circular muscle. 46 Alpha-agonist → Contraction of radial muscle of Iris (Mydriasis). Fear (Sympathetic discharge). Death (Lack of muscular tone due to lack of Ach.) Except opiod intoxication (M-agonist → Pin point Miosis) Alpha-Blocker → Relaxation of radial muscles of Iris (Miosis) Lens: Attached to the ciliary body by ligaments. Ciliary body: that involves - Ciliary epithelium (B2 receptors): responsible for secretion of aqueous humor. - Ciliary muscle (M receptors): responsible for near or far vision. - - Figure 2: The contraction and relaxation of the lens 47  Ciliary Muscle (Muscarinic receptors) M-agonist → Ciliary M. Contraction → Lens contraction → near vision Anti- Muscarinic → Ciliary M. Relaxation → Lens relaxation → far vision Ciliary Epithelium (B2-Receptors) Responsible for secretion of aqueous humor. Contraction of ciliary muscle presses trabecular meshwork → enhancing the flow of aqueous humor through canal of Schlemm. ✓ Ciliary muscle contraction → Increases flow → Decreases IOP. ✓ Ciliary muscle Relaxation → Decreases flow → Increases IOP (Glaucoma). Methods: Place few drops of the agents in the following table into the eyes of rabbits or volunteers and check for the parameters mentioned in the same table, and the results are as follows: parameter Pupil Size Light Accommodation Conjunctival Corneal IOP Reflex Blood vessels sensation Agent Adrenaline ↔ +ve ↔ Pale +ve ↔ Phenylphrine Mydriasis +ve ↔ Pale +ve Inc. Pilocarpine Miosis +ve Near Vision Congestion +ve Dec. Atropin Mydriasis -ve Far Vision Pale +ve Inc. (Congested in High Dose) Xylocaine ↔ +ve ↔ ↔ -ve ↔ procaine ↔ +ve ↔ ↔ +ve ↔ (+ve) indicates the presence of the reflex, (-ve) indicates the absence of the reflex, (↔) indicates that there is no change 48  Adrenaline acts on alpha-receptors causing vasoconstriction of the epithelium of conjunctiva, but it does not cause mydriasis as it cannot be absorbed by the iris. This is also true for procaine (local anesthetic) as the cornea does not absorb it, so it cannot cause loss of corneal reflex. Drug name Action Pupil size Light Corneal Conjunctival Accommodation Palpebral reflex reflex blood fissure vessels Pilocarpine M-agonist myosis normal +ve normal Near vision -ve Tropic-amide Antimuscarinic mydriasis absent +ve normal Far vision -ve phenylephrine α-agonist mydriasis normal +ve Pale No effect +ve proparacaine Local normal normal -ve normal No effect -ve anesthetic Exp. Needs: Atropine, adrenaline, Pilocarpine, Xylocaine, procaine, Phenylephrine, proparacaine, Tropic-amide, oxymetazoline drops, light source and cotton. 49 Lab10: Evaluation of Anti-inflammatory Drugs Pain Pain is an unpleasant sensory and emotional experience Is one way the body tells you something's wrong and needs attention Acute pain typically comes on suddenly and has a limited duration (less than 3 months). It's frequently caused by damage to tissue such as bone, muscle, or organs, and the onset is often accompanied by anxiety or emotional distress. Chronic pain: Defined as ‘Disease of pain’ lasts 3 months or longer than acute pain and is generally somewhat resistant to medical treatment. It's usually associated with a long-term illness, such as osteoarthritis Pain may be NOCICEPTIVE PAIN NEUROPATHIC PAIN MIXED CATEGORY PAIN. NOCICEPTIVE PAIN (Acute pain) Nociceptors Nerves which sense and respond to parts of the body that suffer from damage They signal tissue irritation, impending injury, or actual injury. When activated, they transmit pain signals (via the peripheral nerves as well as the spinal cord) to the brain Time limited, meaning when the tissue damage heals, the pain typically resolves. (Arthritis is a notable exception in that it is not time limited) Tends to respond well to treatment with conventional analgesics 50  Examples include sprains (stretching of ligaments), bone fractures, burns, inflammation. NEUROPATHIC PAIN Result of an injury or malfunction in the peripheral or central nervous system. The pain is often triggered by an injury, but this injury may or may not involve actual damage to the nervous system The pain may persist for months or years beyond the apparent healing (chronic) Less response to treatment with conventional analgesics, but may respond well to other drugs such as anti-seizure and antidepressant medications Examples: reflex sympathetic dystrophy / causalgia (nerve trauma), components of cancer pain, phantom limb pain, and peripheral neuropathy. MIXED CATEGORY PAIN Caused by a complex mixture of nociceptive and neuropathic factors. An initial nervous system dysfunction or injury may trigger the neural release of inflammatory mediators and subsequent neurogenic inflammation. For example, migraine headaches probably represent a mixture of neuropathic and nociceptive pain. Myofascial pain is probably secondary to nociceptive input from the muscles, but the abnormal muscle activity may be the result of neuropathic conditions. Inflammation Associated with injuries, Infections, antibodies, physical injuries. 51  Can be exaggerated response with no apparent benefit Classic symptoms include warmth, pain, redness and swelling Phases: Acute (Injury): Delayed, subacute (Infection) Chronic proliferative phase (Cancer) COX1 Vs COX2 COX-1 and COX-2 convert arachidonic acid to prostaglandin, resulting in pain and inflammation Cyclooxygenase-1 (COX-1) is known to be present in most tissues. In the gastrointestinal tract, COX-1 maintains the normal lining of the stomach. The enzyme is also involved in kidney and platelet function Cyclooxygenase-2 (COX-2) is primarily present at sites of inflammation. Inhibition of COX-1 is undesirable while inhibition of COX-2 is considered desirable COX-2 inhibitors potentially more selective for anti-inflammatory effects Less intestinal bleeding than with nonselective COX inhibitors. NSAIDS NSAIDs are available OTC Analgesic, antipyretic, anti-inflammatory 52  Traditional NSAIDs include aspirin, ibuprofen, naproxen and many other generic and brand name drugs A newer NSAID like celecoxib,"COX-2 inhibitor" or a "COX-2 selective" NSAID Used to relieve pain and reduce signs of inflammation: fever, swelling and redness. Aspirin reduces fever by enhancing cutaneous blood flow and induce sweating and irreversibly inhibitor of inflammatory Cox 2 and PG. NSAIDs also are a common treatment for chronic (long-term) health problems such as arthritis (rheumatoid arthritis, osteoarthritis and others). ASPIRIN AS ANTIPLATELET Aspirin works by irreversibly inhibiting the enzyme cyclo-oxygenase (COX-1) (which is required to make the precursors of thromboxane within platelets), this reduces thromboxane synthesis. Thromboxane is required to facilitate platelet aggregation and to stimulate further platelet activation. Adverse effects  Gastrointestinal (GI) and renal effects. Dyspepsia and upper gastrointestinal adverse events, including bleeding and peptic ulcer.  Reduce renal blood flow and decreasing glomerular filtration by reducing prostaglandin synthesis, thus resulting in salt and water retention through stimulation of RAS. and thereby decrease the efficacy of diuretics, and inhibit the elimination of lithium and methotrexate. 53  On chronic use cause analgesic nephropathy.  Hypocoagulability, which may be serious when combined with other drugs that also decrease blood clotting, such as warfarin  Antagonize the effect of anti-hypertensives, such as ACE Inhibitors, BB, Loop diuretics. part of mechanism of action of these antihypertensive drugs is PG-dependent pathway and inhibition of prostaglandin (PG) synthesis (E1, E2.I2) by NSAIDs lead to reduce the anti-hypertensive effect. EXPERIMENTAL PROTOCOL Methods: - 1- Measure the weight of two mice to determine the dose of drugs. 2- The 1st mouse received normal saline and consider as a control, the 2nd mouse received diclofenac 0.7mg/kg I.P. 3- After 30 minutes of drug administration, the mice were treated with 1% acetic acid at 10ml/kg BW. I.P. 4- After (5) minutes from acetic acid injection, mice were placed in individual cage and the number of abdominal contractions (writhes) was counted for each mouse for a period of 10 minutes. 5- Compare the results between two mice. 5- Calculate the percentage inhibition of writhing as following: % of inhibition = (Wc - Wt) / Wc x 100 54