PHARMACODYNAMICS

1. PHARMACODYNAMICS V. GeršlAccording to: - H.P.Rang, M.M.Dale, J.M.Ritter, P.K.Moore: Pharmacology, 5th ed.- R.A.Howland, M.J.Mycek: Lippincott’s Illustrated Reviews: Pharmacology, 3rd ed.

2. PHARMACOLOGY definition: Study of the manner in which the function of living systems is affected by chemical agents. General pharmacology:describes general actions which determine the action and activity of a drug Pharmacodynamics:mechanisms of the effects of drug on the body Pharmacokinetics:the way the body affects the drug during (with) time (absorption, distribution, metabolism, excretion) Special pharmacology: description of individual drugs and therapeutic groups of drugs

3. Specialisation within pharmacology: as other sciences a wide field was divided in further sub-forms of pharmacology, i.e.: - molecular pharmacology deals (concerns) the effects of drugs from the point of view of their molecular action - pharmacogenetics (genetic aspects of the action) - clinical pharmacology (clinical use and trial of drugs) - toxicology (close to pharmacology) Pharmacotherapy:term very close to P and in fact practical application of P - causal (it is possible to treat the cause of disease: antimicrobial therapy, substitution of some enzymes, hormones ..) - symptomatic (headache...)

4. Schematic representation of drug absorption, distribution, metabolism, and elimination Drug at site of administration Absorption (input) 1 Drug in plasma Distribution 2 Drug in tissues 3 Metabolism Metabolite(s) in tissues Elimination (output) 4 Drug and/or metabolite(s) in urine, feces, or bile (according to Lippincott´s Pharmacology, 2006)

5. PHARMACODYNAMICS evaluate effect of the drug within the body – Phases of drug effects maximum onset diminish lag They depend on pharmacokinetics

6. The main routes of drug administration and elimination Administration Absorption and distribution Elimination Bile Portal system Urine Liver Kidney Metabolites Faeces Oral or rectal Gut Plasma Percutaneous Milk, sweat Skin Breast, sweet glands Intravenous Intramuscular Muscle Brain Placenta Intrathecal CSF Fetus Expired air Inhalation Lung (according to Rang HP, Dale MM et al: Pharmacology, 2003)

7. ROUTES OF DRUG ADMINISTRATION • The route is determined primarily: • - by the properties of the drug (water or lipid solubility, ionization, etc.),- by the therapeutic objectives (e.g. the need of a rapid onset • of action, the need for long-term administration) • - factors associated with the patient • MAJOR ROUTES OF ADMINISTRATION: - enteral, - parenteral

8. Parenteral: IV, IM, SC Commonly used routes of drug administration IV = intravenous IM = intramuscular SC = subcutaneous Sublingual Inhalation Oral Transdermal patch Topical Rectal (according to Lippincott´s Pharmacology, 2006)

9. Variation in oral absorption among different formulations of digoxin. 2 Plasma digoxin concentration (nmol/l) 1 0 0 1 2 3 4 5 Hours (according to Rang HP, Dale MM et al: Pharmacology, 2003)

10. A) ENTERAL :1. O r a l :Giving a drug by mouth is the most common route of administration (though there is the most complicated pathway to the tissues).Some drug are absorbed from the stomach.Major site of absorption and of entry to the systemic circulation is duodenum (it provides a larger absorptive surface).Most drug, absorbed from the GIT, enter the hepatic portal circulation and encounter the liver before they are distributed over body via the general circulation !First-pass effect of the liver means - high metabolism of the drug during its first passage via hepatic circulation and limits often the efficacy of many drugs when taken orally - e.g. propranolol, nitroglycerin. First-pass metabolism by the intestine or liver limits the efficacy of many drugs when taken orally.

11. Ingestion of drugs with food can influence absorption. Food in the stomach - delays gastric emptying  some drugs may be destroyed by acid  limited absorption. Enteric coating of a drug may protect the drug and prevent gastric irritation. Sustained-release tablets - release is prolonged. 2. R e c t a l : 50% of the drainage of the rectal region bypasses the hepatic portal circulationbiotransformation of drugs that are metabolized by the liver is minimized. ! The rectal route has the additional advantage that it prevents the destruction of the drug by intestinal enzymes or by low pH in the stomach. It prevents also the damage of stomach and some other adverse effects of the drug in GIT !

12. First-pass metabolism can occur with orally administered drugs. IV = intravenous Drugs administered IV enter directly into the systemic circulation and have direct access to the rest of body. IV Oral Rest of body Drugs administered orally are first exposed to the liver and may be extensively metabolized before reaching the rest of body. (according to Lippincott´s Pharmacology, 2006)

13. B) PARENTERAL : It is used for drug that are either poorly absorbed from the GIT or for agents (e.g. insulin) that are unstable in the GIT. Used also for the treatment of unconscious patients. The most frequent reason for this administration is the need of the rapid onset of action ! Parenteral administration provides the most control over the actual dose of drug deliveredto the body.

14. 1. I n t r a v e n o u s (IV) : Common parenteral route. For drugs that are not absorbed orally, there is often no other choice. The drug is not absorbed by the GIT first-pass metabolism by the liver is avoided. It permits a rapid effects and maximal degree of control over the circulating levels of the drug. I.v. injection of some drugs may induce hemolysis or other adverse reaction caused by the rapid delivery of high concentrations of drug to the plasma and tissues. The rate of infusion must be carefully controlled (similar concerns apply to intra-arterial administration). ! Risk: the possibility of the most serious and highest adverse effects !

15. 2. I n t r a m u s c u l a r (IM) : Drugs can also be specialized depot preparations - often a suspension of drug in a non-aqueous vehicle (e.g., ethylene glycol, oil). As the vehicle diffuses out of the muscle, the drug precipitates at the site of injection. The drug dissolves slowly  asustained dose over an extended period. E.g. sustained- release protamine zinc insulin, sustained-release haloperidol decanoate - slow diffusion an extended effect. However, i.m. administration is also often used for rapid onset of action(epinephrine in anaphylaxis). 3. S u b c u t a n e o u s (SC) : It provides absorption that is slower. It minimizes the risk associated with intravascular injection. Also may be used solids (e.g., silastic capsules with the contraceptive levonorgestrel - implanted for long-term activity; programmable mechanical pumps can be implanted to deliver insulin in diabetics).

16. 4. S u b l i n g u a l : Placement under the tongue allows the drug to diffuse into the capillary network and therefore to enter the systemic circulation directly (e.g., nitroglycerin). Drug bypasses the liver and is not inactivated by hepatic metabolism 5. I n h a l a t i o n : Used for drugs that are gases (e.g., some anesthetics) or the drugs can be dispersed in an aerosol. Rapid delivery of a drug across the large surface area of the mucous membranes of the respiratory tract and pulmonary epithelium actions can be almost as rapid as i.v. injection. Important e.g. for patients with respiratory complaints (asthma, chronic obstructive pulmonary disease) - drug is delivered directly to the site of action, and systemic side effects are minimized.

17. 6. T o p i c a l : Used when a local effect of the drug is desired. E.g., the treatment of dermatophytosis - clotrimazole is applied as cream directly to the skin; atropine for mydriasis ! It is necessary to take into consideration that a part of the drug used for topical applications may be absorbed (e.g. glucocorticoids) and then affect the whole organism (in case of glucocorticoids for example the mechanisms of the regulation of their secretion) ! 7. T r a n s d e r m a l : This route of administration achieves systemic effects by applications of drug to the skin, usually via a transdermal patch. Used for the sustained delivery of drugs, (e.g., antimotion sickness - scopolamine; antianginal drug - nitroglycerin) The rate of absorption can vary markedly, depending on the physical characteristics of the skin at the site of application.

18. 8. I n t r a n a s a l :Desmopressin (treatment of diabetes insipidus; salmon calcitonin – treatment of osteoporosis – as a nasal spray)Cocaine - the abused drug - by sniffing. 9. Intrathecal / Intraventricularinto cerebrospinal fluide.g. treatment of malignancies – e.g. methotrexate; amphotericin B - cryptococcal meningitis

19. First-pass metabolism can occur with orally administered drugs. IV = intravenous Drugs administered IV enter directly into the systemic circulation and have direct access to the rest of body. IV Oral Rest of body Drugs administered orally are first exposed to the liver and may be extensively metabolized before reaching the rest of body. (according to Lippincott´s Pharmacology, 2006)

20. The main routes of drug administration and elimination Administration Absorption and distribution Elimination Bile Portal system Urine Liver Kidney Metabolites Faeces Oral or rectal Gut Plasma Percutaneous Milk, sweat Skin Breast, sweet glands Intravenous Intramuscular Muscle Brain Placenta Intrathecal CSF Fetus Expired air Inhalation Lung (according to Rang HP, Dale MM et al: Pharmacology, 2003)

21. Changes in physiological functions decrease in functionincrease in function paralysis inhibition - + stimulation excitation

22. HOW DRUGS ACT : MOLECULAR ASPECTS • TARGETS FOR DRUG ACTION • *receptors • * ion channels • * enzymes • * carrier molecules

23. Targets for drug action • A drug = a chemical that affects physiological function in a specific way. • With few exceptions, drugs act on target proteins namely ― enzymes ― carriers ― ion channels ― receptors • Specificity is reciprocal: individual classes of drug bind only to certain targets, and individual targets recognize only certain classes of drug. • No drugs are completely specific in their actions. In many cases, increasing the dose of a drug will cause it to affect targets other than the principal one, and this can lead to side-effects.

24. (according to Rang HP, Dale MM et al: Pharmacology, 2003)

25. Drug specificity: - non specific drug action: Not all drugs act via receptors – e.g., antacids chemically neutralize excess gastric acid. general anaesthetics, osmotic diuretics- act by virtue of their physico-chemical properties. - Interaction with various macromolecules (Na-K-ATPase, inhibition of acetylcholinesterase, as false substrates or inhibitors for certain transport systems or enzymes). - specific (receptors) nonspecific specific according to interactions with targets, effects of drugs are:

26. RECEPTORS • Protein molecules, normally activated by transmitters or hormones. • Many receptors have been cloned. Drug receptor = specialized • macromolecule that binds a drug and mediates its pharmacological • action. • Receptors can be regarded as the sensing elements in the system of • chemical communications that coordinates the function of all the different cells in the body, the chemical messengers being hormone or transmitter substances. • Many synthetic drugs that act either as agonists or antagonists on receptors for endogenous mediators.

27. - specific drug action - most drugs produce effects by acting on specific protein moleculescalled RECEPTORS (they normally respond to endogenous chemicals in the body). Drugs that activate receptors and produce a response are called AGONISTS. Drugs called ANTAGONISTS - combine with receptors, but do not activate them. Antagonists reduce the probability of the agonist combining with the receptor and so reduce or block its action. The activation of receptors is coupled to the physiological or biochemical responses by transduction mechanism that often involve molecules called "SECOND MESSENGERS".

28. k+1 stimulus [R] + [A] [RA] EFFECT k-1 modify factors R = receptor A = drug RA = drug-receptor complex k+1 = constant of association k-1 = constant of dissociation Basic principles The first step of drug action on specific receptors is the formation of a reversible drug-receptor complex, the reactions being governed by the Law of Mass Action – rate of chemical reaction is proportional to the concentrations of reactants:

29. Most drugs exert their effects (both beneficial and harmful) by interacting with receptors.Receptor = specialized target macromolecules-present on the cell surface or intracellularly. Receptors bind drugs and mediate their pharmacologic actions.Drug + Receptor  Drug-receptor complex → Biologic effect The formation of the drug-receptor complex  biologic response; the magnitude of the response is proportional to the number of drug-receptor complexes.Receptor not only has the ability to recognize a ligand (drug), but can also couple or transduce this binding into a response by causing a conformational change or a biochemical effect.

30. Interaction of receptors with ligands - formation of chemical bonds (mostly electrostatic and hydrogen bonds and van der Waals forces) – i.e., mostly noncovalent bonds (covalent are important in toxicology mostly). The bonds are usually reversible. The interaction between a drug and the binding site of the receptor depends on the complementarity of "fit" of the two molecules (the key and the lock). The precise fit required of the ligand echoes the characteristics of the "key,„; the opening of the "lock" reflects the activation of the receptor. The closer the fit and the greater the number of bonds - the stronger are attractive forces between them - the higher the affinity of the drug for the receptor. The ability of a drug to combine with one particular type of receptor is called specifity. No drug is truly specific but many have a relatively selective action on one type of receptor.

31. MAJOR RECEPTOR FAMILIES Mostly proteins that are responsible for transducing extracellular signals into intracellular responses. 1) ligand-gated ion channels 2) G protein-coupled receptors 3) enzyme-Iinked receptors 4) intracellular receptors. (Note: Pharmacology defines a receptor as any biologic molecule to which a drug binds and produces a measurable response. I.e., enzymes and structural proteins can be considered to be „pharmacologic receptors“) .

32. (according to Rang HP, Dale MM et al: Pharmacology, 2003)

33. General structure of four receptor families. N A Type 1 Ligand-gated ion channels (ionotropic receptors) Binding domain C x 4 or 5 Channel lining B Type 2 G-protein- coupled receptors (metabotropic receptors) Binding domains N C G-protein coupling domain N Binding domain C Type 3 Kinase-linked receptors Catalytic domain C C Binding domain D Type 4 Nuclear receptors DNA-binding domain („zinc fingers“) N (according to Rang HP, Dale MM et al: Pharmacology, 2003)

34. Transmembrane signaling mechanisms. ALigand-gated ion channels Example: Cholinergic nicotinic receptors BG protein-coupled receptors Example: a and b adrenoreceptors CEnzyme-linked receptors Example: Insulin receptors DIntracellular receptors Example: Steroid receptors g b a Ions R-PO4 R Protein and receptor phosphorylation Protein phospho-rylation Protein phosphorylation and altered gene expression Changes in membrane potential or ionic concentration within cell INTRACELLULAR EFFECTS (according to Lippincott´s Pharmacology, 2006)

35. Ligand-gated ion channels • Called ionotropic receptors. • Responsible for regulation of the flow of ions across cell membranes • Involved mainly in fast synaptic transmission. • Several structural families, the commonest being heteromeric assemblies of 4 - 5 subunits, with transmembrane helices arranged around a central aqueous channel. • Ligand binding and channel opening occur on a milliseconds. Response to these receptors is very rapid. • Examples: nicotinic acetylcholine, gamma-aminobutyric acid type A (GABAA) and 5-hydroxytryptamine type 3 (5-HT 3) receptors.

36. G-protein-coupled receptors (metabotropic receptors). • Peptide withseven membrane-spanning regions - receptors are linked to a G protein(s). One of the intracellular loops is larger than the others and interacts with the G-protein. • The G-protein = membrane protein - 3 subunits (α,,γ), α-subunit binds GTP,possessing GTPase activity. Binding of the ligand to the extracellular region of the receptor activates the G protein GTP replaces GDP on the α-subunit. • Dissociation of the G protein  both the α-GTP and the ,γ subunit interact with other cellular effectors – so called second messengers (responsible for further actions in the cell). • There are several types of G-protein, which interact with different receptors and control different effectors. • Examples: muscarinic acetylcholine receptor, adrenoceptors and neuropeptide receptors. Responses last several seconds to minutes.

37. Extracellular space Hormone of neurotransmitter The recognition of chemical signals by G-protein coupled membrane receptors triggers an increase (or, less often, a decrease) in the activity of adenylyl cyclase. Cell membrane 1 – Unoccupied receptor does not interact with Gs protein g b Receptor a GDP Gs protein with bound GDP Inactive adenylyl cyclase Cytosol 2 – Occupied receptor changes shape and interacts with Gs protein. Gs protein releases GDP and binds GTP. g b a GTP Inactive adenylyl cyclase GTP GDP 3 – a Subunit of Gs protein dissociates and activates adenylyl cyclase. g b ATP a GTP Active adenylyl cyclase cAMP + PPi 4 – When hormone is no longer present, the receptor reverts to its resting state. GTP on the a subunit is hydrolyzed to GDP, and adenylyl cyclase is deactivated. g b a (according to Lippincott´s Pharmacology, 2006) GDP Inactive adenylyl cyclase Pi

38. Effectors controlled by G-proteins Two key pathways are controlled by receptors, via G-proteins. • Adenylate cyclase (AC)/cAMP: • ― AC catalyses formation of the intracellular messenger cAMP • ― cAMP activates various protein kinases, which control cell function in many ways byphosphorylation of enzymes, carriers and other proteins. • Phospholipase C/inositol trisphosphate (IP3)/diacylglycerol (DAG) • ― catalyses the formation of two intracellular messengers, IP3 and DAG, from • membrane phospholipid • ― IP3 acts to increase free cytosolic Ca2+ by releasing Ca2+ from intracellular • compartments • ― increased free Ca2+ initiates e.g. contraction, secretion, enzyme activation,membrane hyperpolarisation • ― DAG activates protein kinase C – control of many cellular functions by • phosphorylating of proteins. • Receptor-linked G-proteins also control: ― phospholipase A ( formation of arachidonic acid and eicosanoids) • ― ion channels (e.g. K and Ca channels  affect membrane excitability, transmitter release, contractility, etc.).

39. Enzyme-Iinked receptors (Kinase-linked receptors) They have a cytosolic enzyme activity as an integral component of their structure or function. Binding of a ligand to an extracellular domain activation or inhibition of this cytosolic enzyme activity. Duration of responses - minutes to hours. The most common are those that have a tyrosine kinase activity as part of their structure. Binding of a ligand activates the kinase  phosphorylation of tyrosine residues of specific proteins. E.g., when insulin binds two receptor molecules, their intrinsic tyrosine kinase activity causes autophosphorylation of the receptor itself. In turn, the phosphorylated receptor phosphorylates target molecules-insulin-receptor substrate peptides-that subsequently activate other important cellular signals (e.g., IP3, and the mitogen-activated protein kinase system).

40. Receptors for various hormones (e.g. insulin) and growth factors incorporate tyrosine kinase in their intracellular domain. • Cytokine receptors- intracellular domain binds and activates cytosolic kinases when the receptor is occupied. • A common architecture - large extracellular ligand-binding domain connected to the intracellular domain. • Signal transduction - dimerisation of receptors, followed by autophosphorylation of tyrosine residues. The phosphotyrosine residues act as acceptors for the SH2domains of a variety of intracellular proteins - control of many cell functions. • Involved in controlling cell growth and differentiation; also act indirectly by regulating gene transcription. • Important pathways: • ― Ras/Raf/MAP kinase pathway –cell division, growth and differentiation • ― Jak/Stat pathway - activated by many cytokines; it controls the synthesis and release of many inflammatory mediators.

41. Protein phosphorylation in signal transduction • Many receptor-mediated events involve protein phosphorylation, which controls the functioning and binding properties of intracellular proteins. • Receptor-linked tyrosine kinases, cyclic nucleotide-activated tyrosine kinases, and intracellular serine/threonine kinases comprise a „kinase cascade“ mechanism that leads to amplification of receptor-mediated events. • There are many kinases, with differing substrate specificities, allowing specificity in the pathways activated by different hormones.

42. Intracellular receptors The receptor is intracellular ligand must diffuse into the cell to interact with the receptor. E.g., steroid and thyroid hormones, vitamin D. Activated ligand-receptor complex migrates to the nucleus, where it binds to DNA sequences  regulation of gene expression. Activation and response of receptors is longer - cellular responses delayed (30 minutes or more); the duration of the response (hours to days) is much greater than that of other receptors.

43. Receptors that control gene transcription (nuclear receptors) - Intracellular receptors • Ligands include steroid hormones, thyroid hormones, vitamin D and retinoic acid, certain lipid-Iowering and antidiabetic drugs. • Receptors are intracellular proteins, so ligands must first enter cells. • DNA-binding domain attached tovariable control domains. • DNA-binding domain  promotion or repression of particular genes. • Effects are produced as a result of altered protein synthesis slow in onset.

44. A lipid-soluble drug diffuses across cell membrane and moves to the nucleus of the cell Mechanism of intracellular receptors Drug Drug TARGET CELL CYTOSOL Drug Inactive receptor NUCLEUS Activated receptor complex The drug binds to an intracellular receptor. Gene The drug-receptor complex binds to chromatin, activating the transcription of specific genes. mRNA mRNA Specific proteins (according to Lippincott´s Pharmacology, 2006) Biologic effects

45. Transmitter substances - chemicals released from nerve terminals - they bind to the receptors - this triggers post-synaptic events effects (e.g., muscle contraction or glandular secretion). Then the transmitter is inactivated (by enzymic degradation or reuptake). Hormones - chemicals released into the bloodstream, they produce physiological effects on tissues - specific hormone receptors. Drugs may interact with the endocrine system by inhibiting or increasing hormone release or by activation (e.g., steroidal anti-inflammatory drugs), or blockade (e.g., oestrogen antagonists) of the receptors. Local hormones(autacoids) - histamine, serotonin, kinins, prostaglandins - released in pathological processes. Enzymes Catalytic proteins - they increase the rate of chemical reactions. Some drugs act by inhibiting enzymes (anticholinesterases, MAO inhibitors, inhibitors of COX).

46. SECOND MESSENGERS • Chemicals whose intracellular concentration increases (ordecreases) • response to receptor activation by agonists and triggerprocesses that • may result in a cellular response. • The most studied: Ca ions, cAMP, inositol-1,4,5-triphosphate (IP3), • diacylglycerol (DG) • cAMP - formed from ATP by adenyl cyclase (e.g. when -adrenoceptors • are stimulated). cAMP activates protein kinase A whichphosphorylates • a protein (enzyme or ion channel) leading toan effect. • IP3 and DG- formed from membrane by activation of a phospholipase C. • All messengerscan activate kinases, but IP3 does this indirectly by • mobilizing intracellular calcium stores.

47. DOSE-RESPONSE RELATIONSHIPS Agonist = agent that can bind to a receptor and elicit a response. The magnitude of the drug effect depends on its concentration at the receptor site, which in turn is determined by the dose of drug administered and by factors characteristic of the drug (e.g. rate of absorption, distribution, metabolism).

48. Semilogaritmic DRC - effect of the dose on the magnitude of pharmacologic response. 100 Drug A Drug B 50 Percentage of maximum effect EC50 0 log [Drug] The potency of drug can be compared using the EC50, the smaller the EC50 the more potent the drug. (according to Lippincott´s Pharmacology, 2006)

49. Graded dose-response relations (DRC) As the concentration of a drug increases, the magnitude of its effect also increases. The relationship between dose and response is a continuous one, and can be described for many systems by application of the law of mass action. The response is a graded effect, i.e., the response is continuous and gradual. Relationship between dose and response(effect). Few exceptions: a quantal response - describes all-or-nothingresponse. A graph of the relationship is known as a graded dose-response curve. (plotting the magnitude of the response against increasing doses of a drug) - a rectangular hyperbola. Two important properties of drugs can be determined by graded DRC: 1/ Potency (affinity), a measure of the amount of drug necessary to produce an effect of a given magnitude. 2/ Efficacy of the drug.

50. The effect of drug - analysed by plotting the magnitude of the response versus the log of the drug dose - graded DRC. Relationship between dose and response(effect). It can be evaluated by a dose-response curve (DRC). Graded dose-response curve is given by plotting the magnitude of the effect (ordinate) against the log of the drug dose or concentration (abscissa). Agonist =a drug capable of fully activating the effectors when it binds to the receptor.