pharmacodynamics.pdf

PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE Block: Foundations Block Director: James Proffitt, PhD Session Date: Monday, August 5, 2024 Time: 10:00 am – 12:00 pm Instructor: Todd Vanderah, PhD Department:...

PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE Block: Foundations Block Director: James Proffitt, PhD Session Date: Monday, August 5, 2024 Time: 10:00 am – 12:00 pm Instructor: Todd Vanderah, PhD Department: Pharmacology Email: [email protected] INSTRUCTIONAL METHODS Primary Method: IM13: Lecture ☐ Flipped Session ☐ Clinical Correlations Resource Types: RE18: Written or Visual Media (or Digital Equivalent) INSTRUCTIONS Please read lecture objectives and notes prior to attending session. READINGS RECOMMENDED Reading: Chapters 2-3. Katzung BG & Vanderah TW. Basic & Clinical Pharmacology, 15ed, (2021). McGraw-Hill Medical. [AccessMedicine] Quarterly Journal of Medicine, New Series 83, No. 301, pp. 339- 353, May 1992: Barnes PJ. Molecular biology of receptors. LEARNING OBJECTIVES A. Drug Receptors & Signal Transduction 1. Define the terms Pharmacodynamics and Pharmacotherapeutics. 2. Describe the chemical nature of receptors. 3. Explain the chemical interaction between drugs and receptors and what it takes for a drug to have affinity for a receptor. 4. Give evidence for the existence of receptors. 5. Describe the components of drug receptor systems. 6. Define agonist, partial agonist and antagonist drugs in terms of affinity and efficacy. 7. Define potency, efficacy, competitive antagonist and noncompetitive antagonist. 8. Define signal transduction. 9. Describe (draw out) Gs, Gi and Gq protein coupling. 10. Describe how a G-protein can influence gene transcription. 11. Describe how a G-protein can alter ion channel activity. 12. Describe how kinase receptors result in signal transduction. 13. Describe the mechanism of corticosteroid receptors. B. Dose-Response Curves 14. Be able to plot data on a typical log dose-response curve. 15. Understand how dose-response curves can be used to determine potency, efficacy, and the nature of drug interactions. 16. Define graded and quantal dose-response curves. Block: Foundations | VANDERAH [1 of 24]  PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE 17. Explain why potency is often expressed as D50 or ED50. 18. Compare and contrast “therapeutic window” and "therapeutic index", and know how to calculate both. CURRICULAR CONNECTIONS Below are the competencies, educational program objectives (EPOs), block goals, disciplines and threads that most accurately describe the connection of this session to the curriculum. Related Related Competency\EPO Disciplines Threads COs LOs CO-01 LO-01 - 13 MK-01: Core of basic sciences Pharmacology H & I: Acute Care CO-01 LO-14 - 16 MK-01: Core of basic sciences Pharmacology H & I: Acute Care CO-02 LO-16 - 18 MK-06: The foundations of Pharmacology H & I: Acute therapeutic intervention, including Care concepts of outcomes, treatments, and prevention, and their relationships to specific disease processes CONTEXT: In order for drugs to have an effect on cellular targets they must interact with cellular proteins (i.e., receptors, enzymes), that may rapidly change ion concentrations, phosphorylation state or initiate a pathway of signal transduction, which in turn may cause a change in gene expression by the cell. It’s important to note that cell receptor and signal transduction pathways are critical in regulating all aspects of cellular function, not just drug action. In this lecture we focus on the signal transduction pathways that are most commonly used by drugs. You should review the content on Cell Signaling for background. INTRODUCTION Pharmacology is the science basic to medicine that is about the effects of chemicals on living systems at all levels of organization (molecular to the whole body). The chemicals may be drugs used to prevent, diagnose or treat disease. Drugs modify physiological processes-they do not create new processes or effects. The relationship between the dose of a drug given to a patient and the utility of that drug in treating that patient’s disease is described by a drug’s pharmacokinetics and pharmacodynamics. Pharmacodynamics deals with the study of the biochemical and physiological effects of drugs and their mechanisms of action. Pharmacokinetics is thought of as the body having actions on the drug Block: Foundations | VANDERAH [2 of 24] PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE whereas pharmacodynamics is thought of the drug having effects on the body. Pharmacogenomics the relationship of the individual’s genetic makeup to their response to specific drugs. This area entails the study of genetic factors that underlie variation in drug response. Pharmacotherapeutics is the use of drugs in the prevention and treatment of disease. Many drugs stimulate or depress biochemical or physiological function in human beings in a sufficiently reproducible manner to provide relief of symptoms or, ideally, to alter favorably the course of disease. Conversely, chemotherapeutic agents are useful in therapy because they have minimal effects on human beings but can destroy or eliminate pathogenic cells and organisms. What is important to remember is that virtually all drugs result in more than one effect. In general, one effect predominates over a particular dose range, i.e., the therapeutic window and, within this dose range the drug may be termed selective. If a drug resulted in one and only one effect the drug would be termed specific. It is the goal of pharmacotherapeutics to achieve specificity, however that goal is often unachievable. Toxicity may result if the dose range is exceeded. It has often been emphasized that there is only a quantitative difference between a drug and a poison. One of the characteristics of drug actions is a direct relationship between dose administered and the degree of response obtained. Small doses of drugs produce small effects, larger doses produce larger effects, and very large doses may produce toxic effects. Paracelsus observed that "the difference between a drug and a poison is the dose," in recognition of frequent toxic effects of large doses of drugs. Remember different drugs will vary on their ability to produce an effect. This concept is termed drug efficacy, something we will discuss later in the chapter. Drugs may produce their biological effects by means of different actions: at enzyme active sites, on nucleic acids, on membrane protein structures, on transport mechanisms, on gene transcription, or on ion channels. One very important mechanism that applies to many drugs, hormones and neurotransmitter chemicals is ability to induce biological effects by actions on specific receptors. DRUG-RECEPTOR INTERACTIONS The onset, intensity and duration of responses to chemicals are determined by factors that govern chemical concentration at the site of action. In addition, in the great majority of cases, the drug molecule interacts with a selective molecule in the biological system that plays a regulatory role, i.e., a receptor molecule. In order for a drug to interact with its receptor, a drug molecule must have the appropriate size, electrical charge, shape and atomic composition (Figure 1). Block: Foundations | VANDERAH [3 of 24] PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE A drug’s “attractiveness” to a receptor is referred to as affinity. Receptors contain specific recognition or binding sites linked to cellular regulatory processes. The recognition sites exhibit specific patterns of chemical forces that complement those of the drug. As the drug molecule approaches the receptor recognition site, the complementary chemical forces create mutual attraction between the drug and receptor to allow binding. Binding is usually weak and easily reversible. The binding forces include ionic bonds (complementary charges), hydrogen bonds and van der Waals forces. Stronger binding (irreversible) includes covalent bonds. The complementary forces are precisely arranged in space so that opposite charges or other attractive forces between the drug and receptor show spatial complementarities (illustrated in Figure 1). Figure 1. Conceptual diagram of the interaction between a drug (neurotransmitter) molecule and a drug receptor. The drug and the receptor show chemical and spatial complementarily providing mutual attraction. The drug binds to the receptor usually by means of weak ionic bonds, hydrogen bonds and van der Waals forces. After binding of an agonist, changes in membrane function or receptor conformational folding may occur, illustrated as a change in” shape” of the receptor macromolecule. An antagonist will typically bind to the receptor without change in receptor shape but block the binding pocket from other drugs interacting. Because the drug-receptor binding forces are relatively weak (unless there are covalent bonds made, irreversible), the drug-receptor complex easily dissociates (reversible interaction). After the drug molecule interacts with the receptor, changes in distribution of chemical charges, changes in folding of the receptor protein, and/or change in drug receptor conformation occur to activate the intracellular transducer mechanism to which the receptor is coupled. Because of the relatively weak forces (excluding covalent bonds which are not often found/preferred in the design of medications) that bind the drug to the receptor molecule, the interaction usually is easily reversible and the drug molecule is not permanently changed as a result of the receptor binding (in contrast to enzyme-substrate interactions). Block: Foundations | VANDERAH [4 of 24] PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE RECEPTOR ELEMENTS Some drug receptors consist of three interrelated components: (1) a recognition site that binds the drug molecule, (2) a transducer mechanism that translates binding into a biochemical change, and (3) an amplification mechanism that results in alteration of cellular function. The alteration may be excitatory, to increase cellular function (contraction of smooth muscle, increase in secretion by a gland cell), or inhibitory, to decrease cell function (hyperpolarization of nerve cell, relaxation of smooth muscle). The elements of the receptor mechanism are illustrated schematically in Figures 2 & 3. Figure 2. Components of a drug receptor system. The recognition site (binding site) recognizes the drug and binds it. After drug binding, transducer mechanisms convert the message into a biochemical event. Some receptors are ion channels allowing ions to flow either in or out of the cell depending on the receptor activated. In some receptor systems, guanine nucleotide (GTP) binding proteins (G proteins) serve as part of the transducer mechanism, in other systems the receptor plays a role phosphorylation (kinase receptors). Finally, an amplification system multiplies the receptor effect through activation of enzymes (adenylate cyclase, phospholipase C, kinases or other enzymes). These can include the indirect opening or closing of ion channels, or by other mechanisms that change function of the cell termed "response" (also see Figure 3). Drugs that interact with specific receptors and cause a change in conformation of the receptor, transduction (whether receptor activation results in an increase or a decrease in cell function) and a change in amplification are known as agonists. Drugs that block access of an agonist to its receptor are known as antagonists (do not change receptor conformation, transduction) (Table 1). Table 1. Summary of drug-receptor terminology. Agonist Interactions with receptor recognition site to induce change in cell function (increase or decrease). Antagonist Interacts with receptor recognition site but does not itself induce change in cell function, only blocks access of agonist (exogenous or endogenous) to receptor site. Partial Agonist Interacts with receptor recognition site but a partial agonist cannot induce a maximum response (reduced efficacy) Affinity Ability and attraction of drug-receptor interaction Block: Foundations | VANDERAH [5 of 24] PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE Intrinsic Activity Ability of a drug to induce intracellular activity (activate intracellular proteins) Efficacy The measure of a drug’s ability to induce a response by itself (i.e., antagonist has no efficacy) Potency Dosage (concentration) required to induce, or block a response DRUG RECEPTORS Receptors are single macromolecules or aggregates of macromolecules, proteins (or glycoproteins), located in cell plasma membranes or in the cell cytoplasm that interact with endogenous substances (neurotransmitters, cytokines, chemokines, prostaglandins, growth factors, hormones, etc.) or exogenous substances (drugs or toxins). Neurotransmitters and peptide hormones generally act at receptors located on the cell surface whereas steroid hormones (which are more lipid soluble) act on intracellular receptors. Enzyme modulators act at receptors located either in cell plasma membranes (some kinases) or within the cytoplasm (cyclooxygenase enzymes, COX). Receptors recognize specific messenger chemicals and, when activated, alter some cellular process that causes specific biochemical events in the cell to alter function. Thus, activation of a specific receptor in a specific cell type leads to a specific series of biochemical events in that cell. The same receptor in a different cell may lead to a different biochemical event. The receptors acted upon by drugs are often those responsible for mediating regulatory effects of endogenous neurotransmitters and hormones. The drug targets on mammalian cells (Fig. 3) can be broadly divided into four categories of receptors: 1) Ligand Gated Ion Channels (inotropic), 2) G-Protein Coupled Receptors (GPCRs), 3) Kinase Receptors, and 4) Nuclear Receptors. 2, 3 and 4 are termed Second Messenger Receptors (metabotropic). 5 th and 6th categories of receptors are enzymes and transporters. Enzymes, typically located within the cytoplasm of cells, are also considered receptors and will be discussed in more detail in the drug metabolism chapter. Transporters, located within plasma membranes, function similar to ion channels except that they transport larger molecules across membranes (i.e., P-glycoproteins). Block: Foundations | VANDERAH [6 of 24]  PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE Figure 3. Categories of Receptors and their Effectors receptors Metabotropic receptors Pharmacology, Rang et al., 5th ed. 2003. Evidence for the existence of drug receptors comes from several types of observations. (1) The extreme potency of some drugs suggests the presence of specific sites of interaction in cells. Such small amounts of drug producing very large effects suggest a mechanism on cells (receptors) that amplify effects. For example, lysergic acid diethylamide (LSD) in a dose of only 1 µg/kg taken orally can produce profound effects on thought and behavior (based on my readings...), even though most of the drug never even reaches the critical nerve cells in the brain. (2) Similar molecules often produce similar effects, suggesting interaction with a specific structure within the body. For example, norepinephrine and epinephrine produce many similar biological effects even though they are slightly different in chemical structure. (3) Stereoisomers usually differ in pharmacological activity or potency, even though they have essentially identical physical and chemical properties. In other words with stereoisomers only one isomer will fit the receptor and produce an effect whereas the other isomer will not fit and will not produce an effect. For example, dextrorphan is very, very weak as an opioid analgesic than the active stereoisomer, levorphanol. Dextrorphan does not fit the receptor and can be sold over the counter since it is not considered to act at the opioid receptor (non-narcotic). Whereas levorphanol can fit the opioid receptor, is a narcotic and cannot be sold over the counter. The difference in activity of stereoisomers suggests very precise steric interactions between drug and receptor. (note; dextrorphan is very similar to dextromethorphan found in cold medications) (4) Competitive antagonists can specifically block the actions of individual drugs, hormones or neurotransmitters without blocking others. For example, atropine selectively blocks certain responses to acetylcholine without affecting responses to norepinephrine or other neurotransmitters. Studies with antagonists indicate the presence of numerous types of distinct receptors, many of which may be present in a single cell. (5) Biochemical Block: Foundations | VANDERAH [7 of 24] PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE radioligand binding studies provide evidence for specific sites that selectively bind certain drugs, hormones and neurotransmitters. Similar molecules can be shown to compete for the same binding sites. (6) Cloning; Several receptors have been isolated, purified, cloned, reconstituted and transfected into cell lines in order to further study their function. Exciting progress in the cloning of receptors has led to the discovery of many receptors including receptors with unknown function resulting in the receptors being labeled as “orphans”. ION CHANNELS Some membrane receptors are ion channels (ligand gated channels) (ionotropic). The drug receptor interaction results in a change in receptor protein conformation allowing ions to flow either in or out of the cell. The nicotinic cholinergic receptor found on skeletal muscle, for example, is an ion channel. When acetylcholine binds to this nicotinic receptor, the receptor/channel opens and allows cations (Na + and Ca+2) to flow into the skeletal muscle cell resulting in the depolarization and ultimately muscle contraction. (Fig 3.1). Keep in mind that acetylcholine does not only act at nicotinic ion channels but can also interact at muscarinic G-proteins. The function of a neurotransmitter-receptor interaction is dependent on what receptor it is interacting with and in what cell type (not all cells express all receptors- this brings the diversity to pharmacological activity). 2ND MESSENGER RECEPTORS Integral membrane receptors such as the 2nd messenger receptor molecules have been purified and sequenced (metabotropic)(Fig 3. 2-4). They are characterized by having a peptide chain that passes through the cell membrane either multiple times (i.e., G-proteins) or only one time (i.e. kinases). In some cases the receptor may loop through the membrane several times (7 membrane-spanning domains) (Fig. 4). The recognition site often occurs on the extracellular components of the receptors. The inner (cytoplasmic) components are associated with guanine nucleotide- binding proteins (G proteins) (Fig. 4). The G-proteins are in turn coupled to intracellular amplification systems, such as adenylate cyclase, guanylate cyclase, phospholipase C or ion channels. Block: Foundations | VANDERAH [8 of 24] PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE Figure 4. From top left, view clockwise. In the Off position (no agonist binding to receptor) the " subunit of the G-protein is associated with a GDP. Upon agonist (first messenger) binding to the receptor, the receptor changes conformation, associating with the G-protein and the " subunit of the G-protein exchanges the GDP for a GTP. The G-protein (second messenger) is now active resulting in the dissociation from the receptor and other subunits resulting in the interaction with other proteins/enzymes that amplify the drug-receptor message. Eventually the GTP is hydrolyzed to GDP and the G-protein returns to the Off position ready to be re-activated by a drug-receptor complex. G-P ROTEINS AND SIGNAL TRANSDUCTION G-protein receptors are one of the most common receptors and excellent targets for many drugs. When activated by a drug they result in the activation of a signal transduction pathway via an intracellular protein termed, G-protein. G-proteins are actually made up of three separate proteins termed alpha(), beta() and gamma() that normally stay associated with each other (Fig. 4 & 5). In the OFF position of the receptor (no agonist) the  subunit of the G- protein is bound to a molecule of GDP (Fig. 4). Upon agonist binding to the receptor, the receptor undergoes a conformational change in its structure activating the G-protein by exchanging the GDP for a GTP. This exchange event results in the activation of the G protein by causing dissociation of the G-protein from the receptor as well as a dissociation of the  subunit from the  subunits. The  subunit then acting as a second messenger can alter enzyme activity (i.e., adenylate cyclase, phospholipase, etc.) that results in a signal transduction and amplification of the drug-receptor signal. The  subunit can act as a GTPase (the  subunit can hydrolyze GTP to GDP) turning off the signal and re- associating with the  subunit (Fig. 4). There are several different types of G-proteins. Some G-proteins are stimulatory (Gs), some are inhibitory (Gi) at cyclase enzymes (Fig. 5), yet other G proteins (Go, Gq) function to alter phospholipase activity and calcium levels (Fig. 7 & 8). Block: Foundations | VANDERAH [9 of 24] PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE Figure 5. Three different dopamine receptors labeled D 1, D2 and D3 are all G-protein coupled receptors but when dopamine binds to the individual receptors, dopamine can have completely different functions within the cell based on the second messenger that the individual receptor couples with. For example D1 receptors will stimulate adenylate cyclase and D 2 receptors will inhibit adenylate cyclase upon agonist binding. Typically these different receptors D 1 and D2 that oppose each other will NOT be expressed on the same cell. An agonist may interact with a receptor recognition site coupled, for example, by a Gs protein to adenylate cyclase. The agonist would therefore cause increased activity of adenylate cyclase and increased production of cyclic AMP (Fig. 5). Another receptor can be coupled by means of a Gi protein to the adenylate cyclase and activation of the receptor by an agonist would decrease activity of the cyclase (Fig. 5). The intracellular activation continues within the cell. It is well known that the activation of cyclase enzymes can result in the increase in cAMP which has the ability to activate protein kinase A (PKA) as well as bind to proteins that can regulate gene transcription at the level of the nucleus (Fig. 6). G-protein coupled receptors can have multiple quick acting (changes in cAMP and PKA) as well as long term effects (changes in DNA transcription). In addition, the  subunits of the G-protein complex can indirectly gate ion channels from the intracellular side as well as other functions. Block: Foundations | VANDERAH [10 of 24] PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE Figure 6. Drug-receptor complexes can result in the activation of signal transduction pathways that may ultimately lead to protein kinase activity and the alteration in gene transcription at the level of the nucleus. Kinase activity may regulate ion channel function by phosphorylating channels. cAMP activation by G-protein activation may also lead to either an increase in gene transcription and protein production (as above) or could also inhibit certain proteins from being transcribed and translated (not shown). Other families of G-proteins include Gq and Go. The overall function of such Gq-proteins is to increase phospholipase C activity leading to the release of phosphatidylinositol 4,5-bisphosphate (PIP2) into two components, inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 results in the release of intracellular calcium which activates Ca +2 dependent kinases and DAG activates protein kinase C (PKC)(Fig. 7). Block: Foundations | VANDERAH [11 of 24] PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE Figure 7. Neurotransmitter or drug binding to the Gq-protein coupled receptor results in the activation of phospholipase C (PLC) resulting in the activation of IP 3 and DAG, eventual activation of Ca+2 dependent kinases and protein kinase C. Yet, additional G-proteins can activate Phospholipase A2 (PLA2) (Fig. 8). This G-protein coupled pathway results in the release of Arachidonic acid from the cellular membrane. Arachidonic acid is acted upon by enzymes to produce many factors including prostaglandins, thromboxanes and leukotrienes. Figure 8. Transmitter binding to a G- protein coupled receptor resulting in the exchange of GDP for GTP. This exchange results in the activation of phospholipase A2 (PLA2). The activation of PLA2 results in the activation of the Arachidonic Acid pathway. This pathway is important in many physiological conditions and can be blocked in part by steroids, aspirin and NSAIDs acting at the PLA2 level or the cyclooxygenase enzymes (COX). Other second messenger proteins include a very large family of kinase receptors. Agonist interaction with these receptors results in a chain reaction of protein phosphorylation that often results in the modulation of protein function and gene transcription (Fig. 9). Block: Foundations | VANDERAH [12 of 24] PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE Figure 9. Some second messenger receptors only cross the lipid membrane one time and act as a kinase when a drug molecule binds the receptor. When a growth factor binds its receptor the receptor changes conformation, associates with another identical drug-receptor complex and phosphorylates each other. This results in a long line of protein phosphorylation (kinase cascade) that may alter other protein function and gene transcription. Nuclear receptors, which are often found within the cytoplasm, when activated by an agonist will enter into the nucleus and alter gene transcription (Fig. 3.4.). A good example of this type of receptor is the estrogen receptor. In the end, there are hundreds of second messenger proteins amplifying the drug/transmitter effect. These signal transductions via these receptors may result in the activation of several cellular proteins/enzymes that may have anywhere from rapid effects to long lasting changes at the level of the DNA. DOSE RESPONSE CURVES The concentration of drug required to bind to a receptor is a measure of affinity of the drug for the receptor site. Affinity can be measured by in vitro experiments. Drugs that are active in low concentrations have high affinity for the receptors. Drug-receptor interactions usually follow simple mass-action relationships. The greater the number of agonist molecules, the greater the number of receptors occupied at any given moment. For this reason, responses to the drug are graded or dose-dependent (Fig. 10). The magnitude of the response is directly proportional to the fraction of total receptor sites occupied by drug molecules (occupancy theory of drug-receptor interaction). Block: Foundations | VANDERAH [13 of 24] PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE Dose-response relationships are easier to understand when graphed as a dose-response curve. By convention (see Figure 10), dosage is nearly always graphed on the X-axis or abscissa, with the dose increasing from left to right, while the response is graphed on the y-axis or ordinate. Because responses to drugs usually increase in proportion to logarithmic (2-fold, 5-fold, 10-fold) increases in dosage, the dose axis is often expressed on a logarithmic scale. One sometimes sees the term "log dose-response curve" for this reason. Examination of Figure 10 will reveal several characteristics of dose- response curves. As dosage is increased, a point is reached which begins to initiate a response. The lowest dose that just begins to cause a response is the threshold dose. As dosage increases (on a log scale) the shape of the dose- response curve is vaguely similar to the letter "s" and is called a sigmoid shaped curve (Fig. 10). As dosage is increased further, the maximum response is attained and larger doses do not induce a larger response. Note that the middle portion of the dose-response curve is nearly straight; this is known as the linear portion of the dose- response curve. Note also that the dose-response curve has a particular slope. Drugs frequently differ in the slopes of their dose-response curves for a specific effect. Dose-response curves are said to be "flat" or "steep". The most critical and useful portion of the dose-response curve is the linear portion (Between 20% and 80%), specially the point where the response is one-half the maximum response. This point is used for comparison of the potencies of different drugs. For graded dose responses, the dose that produces a 1/2-maximal response is usually known as the ED50 dose (effective dose at 50%) (Figure 10). Figure 10. The Y-axis is the percentage of response of the drug and the X-axis is the dose of drug administered. Elements of dose-response curves; when plotted on a logarithmic scale increasing to the right, most dose-response curves are "sigmoid" in shape. The dose at which the response is one-half the maximum response is termed the ED50, and the dose required to produce the maximum response is the Emax. As the dosage (what actually counts is the concentration of drug in the vicinity of the receptor) increases, an agonist occupies a greater and greater fraction of the total receptor sites and the magnitude of the response increases proportionally until a maximum response to the drug Block: Foundations | VANDERAH [14 of 24] PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE is achieved (Emax, Fig 10). Often, the maximum response occurs when less than 100% of the receptors are occupied, indicating the presence of "spare receptors". With a full agonist, the maximum response attained is usually the maximum effect the receptor/tissue is capable of producing (for example, maximum contraction of smooth muscle). With some agonists, however, their maximum effect is less than that of full agonists and less than the maximum response of the tissue; these agonists are known as partial agonists. Figure 11. The Y-axis is the percentage of response of the drug and the X-axis is the dose of drug administered. The doses required to result in responses to two drugs may differ in themagnitudes of their maximum responses. The partial and full agonists illustrate differences in potency and efficacy among two different drugs. The ability of an agonist to induce a biological response after interaction with a receptor is known as efficacy or intrinsic activity of the agonist (Figure 11). It can thus be said that partial agonists have less efficacy (or intrinsic activity) than full agonists. Even if partial agonists occupy 100% of the receptors, a maximum tissue response is not produced. Tissue amplification (intrinsic activity) can vary depending on the tissue state. For example, the same drug will result in different biological responses in children, versus middle-aged adults, versus the elderly. Likewise drug responses will be different in healthy volunteers versus individuals with diseases, illnesses or habits (i.e., smoking, alcohol, etc.) conducive to changes of the tissue amplification pathway. The amount of drug required to bring about a response is a measure of the drug's potency (Figure 11). A potent drug (agonist or antagonist) is active in low concentrations or doses and is always a reflection of the amount of drug. Potency in vivo can reflect a drug's affinity for receptors and the ability to get to the site of action in tissue or arrive at the receptors (pharmacokinetic characteristics). A full agonist has affinity for its receptor and full efficacy. A partial agonist has affinity for its receptor but is deficient in efficacy. A pure antagonist has affinity for the receptor but NO efficacy. Interactions of drugs with receptors can therefore be explained or described in terms of relative potency and efficacy (Figure 11). Block: Foundations | VANDERAH [15 of 24] PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE ANTAGONISTS Antagonists bind to the recognition site and occupy it, but are not able to activate the transducer component of the receptor system. Antagonists do not have efficacy since they cannot activate the transducer component of the receptor. Therefore, the only action of a pure antagonist is to diminish access of agonist molecules to the receptor. These agonist’s molecules may be endogenous molecules or may be exogenously taken drugs. Antagonists bind to the particular receptor for which they have affinity but, because they lack efficacy, they cannot initiate a response. The antagonist hinders access of agonist to the receptor site of action. In the case of overdose of a drug or when the body itself may make too much of an endogenous molecule an antagonist can block the excess resulting in an observed effect. For example, when one gets very nervous giving a public speech the body naturally may produce more epinephrine (i.e., adrenaline). One way to overcome extreme nervousness is to take a receptor antagonist (i.e., beta-blocker which blocks the epinephrine). However, the effect of a competitive antagonist can be overcome by increasing the concentration of agonist. Because both agonist and antagonist molecules follow the law of mass action in terms of their interactions with receptors, a sufficient increase in the number of agonist molecules will finally allow the agonist to compete with the antagonist molecules for the receptor sites until a fraction of receptors are occupied by the agonist molecules necessary to bring about a maximum response of the tissue (assuming that the agonist is a full agonist). An antagonist that can be overcome by adding more agonist is known as a competitive antagonist (Fig 12, middle panel, Fig 13) (also referred to as reversible). The majority of antagonists used as medications are competitive or reversible antagonists. Some antagonists interact irreversibly with the receptor recognition site (or at times an alternative site that inhibits agonist action), usually by formation of stable covalent chemical bonds between the drug and the receptor. Because agonist molecules cannot act through mass action to compete with the covalently bound antagonist to overcome its blocking action, these irreversible antagonists are known as noncompetitive antagonists (Fig. 12, lower panel). Block: Foundations | VANDERAH [16 of 24] PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE Figure 12. Representative dose-response curves illustrating different drug characteristics and relationships. Top panel. Responses to three agonist drugs. Drug A is more potent than drugs B or C, but drugs B and C are equally potent. Drugs A and B have greater efficacy than drug C. Alternatively, curve A could represent responses to an agonist alone, curves B and C could represent responses to that agonist in the presence of antagonist drugs. Curve B would represent responses of the agonist in the presence of a competitive antagonist, curve C responses of the agonist in the presence of a noncompetitive antagonist. Center panel. Responses to three agonist drugs with equal efficacy but different potencies (A > B > C). Alternatively, curve A could represent responses of an agonist alone, curves B and C responses of the agonist in the presence of different concentrations of competitive antagonist. Bottom panel. Responses to two agonists of somewhat equal potency but different efficacies (A > B). Alternatively, curve A could represent responses of an agonist alone, curve B responses of the agonist in the presence of a noncompetitive antagonist The important point is that the competitive (reversible) antagonist can be “pushed off” the receptor with higher concentrations of an agonist and the dose response curves shift to the right. A noncompetitive (irreversible) antagonist cannot be “pushed off” the receptor and results in a loss of the agonist efficacy. Block: Foundations | VANDERAH [17 of 24] PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE Figure 13. Actual data of how dose-response curves are derived. The panels on the left show actual responses as they appear on the chart recorder. The panel on the right shows the data plotted as dose-response curves. Upper left. Responses of strips of ileum longitudinal muscle in an isolated organ bath to exogenously applied acetylcholine. Middle left. Responses to the same doses of acetylcholine in the presence of neostigmine, which prevents destruction of acetylcholine (i.e., allows acetylcholine to remain in contact with the nicotinic receptor longer resulting in an increased effect). Note that lower doses of acetylcholine are more effective in the presence of neostigmine. Bottom left. Responses to acetylcholine in the presence of atropine, a competitive antagonist at receptors for acetylcholine. Note that higher doses of acetylcholine are required to induce responses, but that the antagonist effects of atropine are reversible (competitive). Right. The data are plotted as three dose-response curves for acetylcholine. The acetylcholine dose-response curve was shifted "to the left" in the presence of neostigmine and shifted "to the right" in the presence of atropine. RESPONSE OF POPULATIONS Another way to measure drug effects is to establish a particular endpoint (i.e., relief of pain, unconsciousness, reduction in blood pressure by 30 mmHg) and determine what dose is required to reach that endpoint in an individual subject. Some subjects will respond to very low doses of the drug, some will require very high doses, but most will be somewhere in the middle. If the number of subjects reaching the preset endpoint at each dose of the drug administered is plotted against the dose, a normal distribution curve will be obtained (Figure 14). Sensitive subjects will fall on the left-hand side of the curve, resistant subjects will be represented on the right-hand side, and most will be clustered in the middle. Sensitivity and resistance are determined by individual differences in drug Block: Foundations | VANDERAH [18 of 24] PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE absorption and distribution, variability in receptor sensitivity, genetic differences, presence of disease, age, and many other factors. The responses obtained with this approach are known as quantal responses, which are all-or-none (in contrast to the graded responses discussed earlier) because the particular endpoint chosen is either produced or not produced at any given dose in any one subject. If the total number of subjects or fraction of the sample population responding by the point any particular dose is reached is plotted against dose (Figure 14), the resulting cumulative frequency distribution curve will be sigmoid in shape, similar to the graded dose-response curve. This type of curve is known as a quantal dose-response curve. Figure 14. Examples of how dose-response curves are obtained from quantal (all-or-none) data. Top panel. Frequency distribution curve obtained for responses of a population sample to different doses of a drug. Most subjects responded at doses of 10 mg/kg or less. Middle panel. Cumulative frequency distribution curve obtained from subjects in the top panel. Note that the ED50 dose (effective dose) corresponds to the peak of the normal distribution curve shown in the top panel. Bottom panel. Cumulative frequency distribution curves for two drug effects: a desired increase in blood pressure (bp) and a toxic cardiac arrhythmia. The ED50 for the increase in blood pressure was 10 mg/kg. The TD50 (toxic dose) for the cardiac arrhythmia was 100 mg/kg. The therapeutic window of the drug is TD50/ED50 = 100/10 = 10. The dose of drug required to produce the endpoint effect in 50% of the subjects is known as the median effective dose and is abbreviated ED50. The median lethal dose (if death is the endpoint) is abbreviated LD50; the median toxic dose (for a particular measure of toxicity) is the TD50. One can similarly express other doses of drug required to affect different percentages of the test population (LD99, ED30, TD10). As no drug produces only a single effect, a different dose-response curve can be constructed for each of its effects (a desired therapeutic effect, a particular toxic effect, death). SAFETY EVALUATION Comparison of two quantal dose-response curves (Figure 14 & 15), one for a desired therapeutic effect and one for a toxic effect can be used to calculate a drug's therapeutic window (minimum effective dose in relationship to the minimum toxic dose). The therapeutic index is a relative measure of safety, calculated as the ratio of the LD50 (lethal dose) to the ED50 for the desired effect (Figure 15). However, some text books Block: Foundations | VANDERAH [19 of 24]  PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE and test questions may refer to the therapeutic index in relationship to the unwanted side effects or toxic dose (TD50). LD50 Therapeutic index = ------------- ED50 A drug with a therapeutic index of 10 would NOT be very good as it would produce death at the higher therapeutic doses. The safest drugs have a large therapeutic index, and it is always nice to have a large therapeutic index. However, often drugs will have unwanted side effects that overlap with the doses for the therapeutic effect. One would like to have drugs with a therapeutic index of 100 or more. Figure 15. Therapeutic Index Therapeutic Index 100 LD50 500 ia = = 250 g es h al at % Response 75 ED50 2 Drugs can harm and even result in death. an de Always seek advice on what drugs to 50 prescribe, start with low doses and be sure to check for drug-drug interactions. Be sure to 25 inform your patients of what to expect (drug effects and SIDE EFFECTS) and make sure 0 0.001 0.01 1.0 10.0 100.0 1000.0 you checkup with your patients after writing a prescription. ED50 (2mg/kg) LD50 (500mg/kg) Log Morphine (mg/kg) Therapeutic 100 ia Window es lg c An a xi 75 To % Response Therapeutic Index 50 25 0 0.001 0.01 1.0 10.0 100.0 1000.0 Log Morphine (mg/kg) Block: Foundations | VANDERAH [20 of 24] PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE Variation in response MUST be anticipated and handled by the individualization of therapy using careful formulation of therapeutic objectives and appropriate monitoring of response. In other words, “HAVE A PLAN”. This is especially important when treating serious illness with drugs whose action is characterized by a steep dose-response relationship, (i.e., a small therapeutic index – dose range between therapeutic effect and the drugs lethal dose) and where the clinical endpoints are indistinct. PRACTICE QUESTIONS - DOSE RESPONSE RELATIONSHIPS For questions 1 - 3, choose the most appropriate answer. 1. Guanine nucleotide-binding proteins (G proteins) are often involved in receptor a. recognition functions b. transduction mechanisms c. amplification mechanisms d. synthesis 2. Responses to drugs acting at receptors may be graded because: a. responses usually can be infinitely large b. responses usually are independent of drug dose c. responses are generally proportional to the fraction of receptors occupied d. the amplitude of the response is proportional to chemical strength of binding between drug and receptor 3. Drug D produced its beneficial effect in 50% of subjects tested at a dose of 2 mg/kg and a severe toxic effect in 50% of subjects at a dose of 30 mg/kg. The therapeutic window of the drug is: a. 0.067 b. 2 c. 15 d. 30 4. When a drug produces a full response we would refer to that drug as being a. a potent drug b. an efficacious drug c. a partial agonist d. an inverse agonist e. a drug with a 1000 fold therapeutic index 5. Drug “Z” results in the right-ward shift of drug “Y”. What type of drug is “Z”? a. a full agonist b. an irreversible agonist c. an inverse agonist d. a potent drug e. a competitive antagonist 6. When a dose response curve shifts to the left we think of the drug as a. more efficacious Block: Foundations | VANDERAH [21 of 24] PHARMACODYNAMICS: DRUG RECEPTORS, SIGNAL TRANSDUCTION & DOSE- RESPONSE b. less efficacious c. more potent d. less potent e. having more affinity for the receptor f. having less affinity for the receptor Bonus question What type of graph/curve would one use to determine if two drugs combined to treat one disease/disorder were better then either drug alone? g. Quantal dose response curve h. Time action curve i. Graded dose response curve j. Dual acting dose response curve k. Isobologram Answers: 1. B 2. C 3. C 4. B 5. E 6. C For Reference Material only Common receptors in Medicine and their coupling mechanisms Dopamine receptors Norepinephrine receptors Serotonin receptors D1 = Gs Alpha1 = Gq 5HT1, 5HT5 = Gi D2 = Gi Alpha2 = Gi 5HT2 = Gq D3 = Gi Beta1, Beta2, Beta3 = Gs 5HT3 = Cation Channel (5 subunits) 5HT4, 5HT6, 5HT7 = Gs ACh Muscarinic receptors M1, M3, M5 = Gq M2, M4 = Gi ACh Nicotinic receptors Cation Channel (5 subunits, see fig.) Glutamate Receptors NMDA, AMPA, Kainate = Cation Channels (4 subunits) mGluR1, mGluR5 = Gq mGluR2, mGluR3 = Gi mGluR4, mGluR6, mGluR7, mGluR8 = Go GABA Receptors GABAA = Anion Channel (5 subunits) GABAB = Gi Block: Foundations | VANDERAH [22 of 24]