GP 3: Second messengers & Factors modifying drug action

Objectives for Self study Receptors – various terminology related to receptors. Receptor Ligand Agonist Antagonist Partial agonist Receptor functions Receptor superfamilies Structure, function & examples for Ligand regulated ion channels GPCR Tyrosinekinase linked receptors DNA linked receptors

First messengers: Endogenous ligands & drugs, acting on GPCR. Second messengers: bring about the cellular response to the first messenger acting on GPCR First messenger Receptor Second messenger Biological activity Most common second messengers: cAMP cGMP Phosphoinositides - Inositol triphosphate (IP3) & Diacylglycerol (DAG)

The second messenger system operates through reversible phosphorylation by phosphokinases (PK). cAMP Adenylyl cyclase (the regulating enzyme) ATP is activated by G protein attached to GPCR cAMP cAMP is hydrolysed by phosphodiesterase (PDE) cAMP stimulates PK which stimulate several other PK. C-AMP has diverse biologic actions – +ve inotropic & chronotropic action on heart. Mobilization of stored energy Calcium homeostasis by parathermone etc.

cGMP Guanylyl cyclase (GC) (the regulating enzyme) Is activated by G protein attached to GPCR. GTP GC cGMP is hydrolysed by PDE. cGMP causes vasodilatation etc.

Phosphoinositides & Calcium: Stimulation of R by agonist Leads to activation of a membrane enzyme ‘phospholipase C’ (PLC) which splits Phosphatidyl-inositol-biphosphate (PIP2) into Diacylglycerol (DAG) and Inositol triphosphate (IP3) . Inositol triphosphate (IP3) diffuses through the cytoplasm to calcium stores and triggers the release of calcium. Calcium binds to calmodulin, which regulates activities of several phosphokinases regulating contraction, secretion, enzyme activation and membrane hyperpolarisation. Diacylglycerol (DAG) is confined to the membrane & activates a calcium sensitive phosphokinase (PK) called PK-C which controls many cellular functions. Action of PK is terminated by dephosphorylation.

RECEPTOR REGUALTION Prolonged drug administration may be associated with certain changes in response to drugs due to regulation of receptor function. Short term regulation (often in minutes) of R occurs through R desensitization or by decreased release of mediator resulting in tachyphylaxis or acute tolerance (gradual ↓ in drug effect) Eg. Ephedrine iv  BP in dog. Response to repeated administration (5 min interval) of ephedrine gradually decreases.

Ephedrine given iv, increases BP in dog indirectly by causing release of norepinephrine from sympathetic nerve endings. Response to repeated administration (5 min interval) of ephedrine decreases gradually due to decrease in the stores of NE available for release. Effect of ephedrine on dog Blood pressure Pressor response to Ephedrine 100 ug. 200 ug.

RECEPTOR REGUALTION Long term regulation occurs through  or  in Receptor expression or number. Upregulation - Prolonged exposure to an antagonist (deprivation of stimulation by agonist) results in  in no. of R which leads to supersensitivity to the agonist. Clinical imp.: Sudden withdrawal of betablocker (propranolol) can precipitate angina or MI. Down regulation - prolonged exposure to agonist results in  in no. of R &  response. Clinical imp.:  response to beta adrenoceptor stimulants (salbutamol) in bronchial asthma.

RECEPTOR REGUALTION Before drug treatment Normal R After chronic agonist R downregulation After chronic antagonist R upregulation

Drug Dose & Response Effect of a drug depends on the dose / concentration of the drug in the system. Dose is the amount of a drug administered at one time or per day in divided doses to produce a pharmacological effect. May be expressed as total quantity of the drug or per unit body weight. Relationship between dose / concentration & effect / response can be expressed graphically through ‘dose-response curves’ (DRC).

Drug B produces effect similar t o drug A, but, in larger doses The dose response curve relates the amount of administered drug to the response. Drug B produces effect similar t o drug A, but, in larger doses Drug A Degree of analgesia Drug B Dose

DRC indicates relation of plasma conc. to response Response  with in dose (The response to a drug is proportional to the number of drug - receptor complexes formed). DRC is a rectangular hyperbola Log-DRC is a sigmoid curve & allows a wide range of drug doses to be plotted on a graph as it expands the scale at lower concentrations & compresses at higher concentrations Log-DRC allows comparison of several different DRC.

DRC & Log DRC %Response %Response Dose Dose

Drug Potency Potency Refers to the amount of drug needed to produce a response. Is indicated by the position of DRC on dose axis. Quantity of drug required to produce 50% of the maximal effect – ED50 – is used to compare the relative efficacy of drugs within the same class. A DRC positioned rightward indicates lower potency & vice versa. Eg. Morphine is more potent than pethidine

Relative potency & Efficacy of drugs 6 Morphine 10 mg. Pethidine 100 mg. Degree of analgesia/ Pain relief score Codeine 60 mg. 10 60 100 Dose in mg.

Maximal efficacy (Efficacy) it is the maximal effect a drug can produce with increasing doses & maximal efficacy is indicated by the height of DRC. Increasing the dose beyond this does not increase efficacy but only increases adverse effects. Clinical significance: Drugs are chosen based on their maximal efficacy & not on potency. Potency determines only the dose.

Therapeutic index (TI) – TI – Toxic dose Eg. 4 or 10 Effective dose 2 2 TD50 or LD50 / ED50 ED - Effective dose; TD - Toxic dose; LD - Lethal dose Drugs with TI of <2 should be used cautiously, as usually patient experiences toxicity along with therapeutic effect. Digoxin, Lithium. Drugs with TI >2, can be used with ease as they are safe at recommended doses. Paracetamol, Penicillin

Emax TD50

Factors affecting action of drugs - Age – pediatric, geriatric - weight - Gender - Pregnancy & lactation - Other diseases - Liver & kidney impairment - Concurrent drug administration – Addition Synergism Antagonism – Chemical Functional Competitive – Reversible Irreversible Noncompetitive - Tolerance - Pharmacogenetics

Factors affecting drug dose & response Age Infants & elderly are more sensitive to drug effects. eg. Morphine Body weight dosage needs to be adjusted for body weight of the patient to achieve effective plasma con. body surface area may be more useful than weight to adjust dose esp. for children & while administering anticancer agents

Gender: Females generally require smaller doses of drugs. Pregnancy: Effect on fetus – Thalidomide – 1960 – promoted as a sedative Susceptiblity to hepatotoxicity - ↑ by Tetracyclines Lactation: Effect on the breast fed baby – Ampicillin rash, laxatives Other diseases: In hypothyroidism - ↓ dose of drugs as body metabolism is slow. Liver & Kidney impairment: ↓ dose of drugs as metabolism and excretion are impaired.

Concurrent drug administration: When 2 or more drugs are given one drug may alter the effect of the other drug resulting in either Additive effect Synergism (Potentiation) Antagonism

Addition: when the combined effect is equal to the sum of the individual effect of both drugs. Paracetamol + Ibuprofen Both are analgesics & show additive analgesic effect. Synergism: when the combined effect is more than the sum of the individual effects of both drugs. Antimicrobial agents given together can have synergistic effect. Ampicillin + Gentamicin – Ampicillin facilitates the entry of gentamicin into the bacteria. Sulphonamide (bacteriostatic) + Trimethoprim (bacteriostatic)  Cotrimoxazole (bactericidal) Act on successive steps in bacterial metabolism with a resultant bactericidal action.

Drug antagonism 1. Chemical antagonism 2. Functional antagonism (physiological antagonism) 3. Competitive antagonism (Pharmacologic antagonism occurring at receptors) a. Equilibrium – reversible b. Non-equilibrium - irreversible 4. Non-competitive antagonism -

Chemical antagonism A chemical antagonist is one that counters the effects of another drug by binding the drug & preventing its action. Eg.: Chelating agent Dimercaprol complexes with mercury & facilitates excretion Protamine & heparin 2. Functional antagonism (physiological antagonism) Both agents act on different receptors & produce effects opposite to each other reducing or canceling the effect of each other. Acetylcholine & Epinephrine Adrenaline & Histamine Insulin & adrenal steroids

3. Competitive antagonism (Pharmacologic antagonism) Agonist & antagonist compete for the same receptor. a. Equilibrium – reversible: the binding is not firm The antagonism  as the con. of the antagonist . The antagonism can be reversed if the concentration of the agonist is . The DRC in the presence of a competitive antagonist will shift towards right & maximal effect will not . Acetylcholine - Atropine Adrenaline– Phentolamine Morphine - Naloxone Agonist Response Antagonist

3. Competitive antagonism b. Non-equilibrium - irreversible – antagonist binds to R firmly. In the presence of non-equilibrium antagonist, the maximum response decreases & the slope of the DRC is shifted downwards. eg. Phenoxybenzamine. 4. Non-competitive antagonism The antagonist acts at a site beyond the R for the agonist & is less specific. It interrupts the receptor-effector linkage. Eg.: Nifedipine blocks the action of vasoconstrictors

Importance of drug antagonism: Treatment of drug side effects – antagonists which block only the unwanted effects of agonist may be used. Eg. Use of atropine with neostigmine in myasthenia gravis Coffee with cold remedies producing sedaton Treatment of drug overdosage – Naloxone for morphine poisoning Protamine for heparin overdosage Treatment of anaphylaxis with adrenaline, a physiological antagonist to histamine. Note: Awareness is imp. to avoid unwanted drug antagonism.

Objectives for self study Second messengers – cAMP, cGMC, IP3. DAG, Calcium Receptor regulation short term & long term Dose response curves, Log DRC Drug potency & maximal efficacy Therapeutic index Factors modifying drug action: Age, weight, gender, physiological states (pregnancy, lactation), other diseases, liver and kidney impairment, Concurrent drug administration (Additive effect, synergism, Drug antagonism)