---start---- pharmacoloy 1/12/98 handout: Receptors and dose-response relationships Fluharty isn't back from his sx; someone else will be filling in but following his outline. receptors - in order for an organism to function, cells must be able to communicate to eachother, and without this ability, the body won't function. cells will secrete a chemical message - hormones, nt, etc - to be received by target cells. there are proteins responsible for accepting the chemical messages, and they are called receptors. receptors are often targeted by drugs. other proteins are also acted upon by drugs. ion channels, enzymes, and carriers/transporters are examples of non-receptor protein classes also targeted by drugs. Pesticides have organophosphates which interfere with cholinesterase - an enzyme. Prozac and cocaine act on carriers and transporter proteins. in toxicology and pharmacology, some people use the word "receptor" to mean any protein that is targeted by a drug - but we're talking about actual receptors in the body that respond to particular chemical messages. the initial concept of receptors as explained in handout was offered in 1905 by Langley. He was struck by the relative specificity and potency of drugs in different tissues, and figured that there must be something in some cells that allowed them to interact with a particular drug. The kinds of chemicals that act on receptors are collectively called "ligands" and these are molecules that bind receptors - endogenous as well as exogenous. These compounds can occupy the receptor and then initiate a series of cellular responses (agonists), or they can bind the receptor and not initiation a response (antagonists). within the receptor family are four superfamilies: ion channel receptors G protein coupled receptors growth hormone receptors steroid/thyroid hormone receptors receptors are proteins that serve a bifunctional role - 1. they bind agonist (recognition), 2. they initiate a cellular response to the agonist (effector action). the superfamiilies are organized based on the architecture and mechanisms of cellular response. the first three are all membrane bound proteins which are in the cell membrane - part of the protein binds the ligand (agonist) and part initiates the effect. eg - in growth hormone receptor, the intracellular part initiates response. the steroid/thyroid hormone receptors are cytosolic proteins - not membrane bound. the receptor then floats into the nuclear compartment to regulate gene regulation. we're going to focus on ion channels and g protein receptors. fig 2 in handout is showing the nicotinic ACH receptor - note it is a membrane bound ion channel. the extracellular side binds ACH in specific binding sites. remember in ECF is greater concentration of Na+ than in ICF. the electrostatic gradient is into the cell, but the channel doesn't open up until the binding sites are full of ACH. The time from when ACH hits the receptor until effect occurs is very small because the receptor is the effector as well. this makes ion channels good for neurons and so forth, for when you need rapid response. g protein coupled receptor - fig 1 in handout the extracellular part is on top, the intracellular part is on the bottom. there are a number of transmembrane domains as well. the binding of the ligand (agonist) here tends to be on the extracellular part and the tops of the transmembrane domains. the intracellular part acts with cellular components to initiate the response. since this protein isn't the effector protein, it needs help to communicate to the cell that the ligand has bound. so it gets help from a transducing protein called a G protein, which works by coupling with the intracellular domain, receiving a signal, and then uncoupling and initiating a response. the handout has details on this. fig 3 in handout regardless of actual cellular cascade for this class of receptors there are some common attributes for the way it works - there are receptor molecules in the cell membrane. the ligand binds the extracellular part of the receptor - this is the first message. that binding is communicated to an associated intracellular G protein which is coupled tightly to the receptor. the G protein then dissociates off the receptor and floats over to an enzyme (phospholipase C) which is the amplifying enzyme. phospholipase C takes inositol polyphosphate and cleaves it into two second messengers - IP3 and diacylglycerol - the second messengers. these then go to effector enzymes themselves - eg DAG goes to phosphokinase C which has long duration effects on cell functions, and IP3 causes calcium release, which activates calcium dependent kinases, which have short term effects. SO, G protein activates the phospholipase C, which makes second messengers, which work on other cellular components to initiate cellular response. cAMP mediated response: receptor connected to G protein - message 1 binds receptor, transducing G protein goes over and activates adenylate cyclase, which takes ATP and makes it into cAMP (2nd messenger) which then works on other proteins such as protein kinase A, which has functional effects on the cell. so, with these G protein coupled receptors, response is much slower - many more steps are occuring. The time from ligand binding to cellular response can be seconds. growth hormone receptors take minutes to cause cellular response steroid receptors act directly on gene regulation and transcription, so it can take hours to see the response. how can a G protein receptor modulate an ion channel - fig 4 in handout. see the K+ channel that is open on the top right. there is a receptor coupled with a G protein - it binds serotonin, the G protein transduces the signal, activates adenylate cyclase (amplifying enzyme), makes cAMP (second messenger) cAMP acts on protein kinase A to modulate the activity of the K+ channel - makes it close. so it's the same kind of system. How do we study receptors? three main ways. Assay techniques: bioassay - involves having an isolated tissue preparation, which has a response to a particular ligand. you can use the response to see how much of the factor you are studying is present and how potent the effect of the drug is. we're looking at biological activity of a drug in this test. We can look at purity of a sample for quality control eg of insulin, steroids, heparin. even though we can produce drugs via recombinant methods these days, having something be 100% pure doesn't always mean it is 100% biologically active. example - recently a lab purchased some radiolabelled angiotensin II. It cost them $4000. They then tried to use it and the experiments all failed. Bottom line - it took three weeks to figure out that the AT II was only 50% active even though it was 100% pure. so bioassays are very important to quantitate biological activity. radioligand binding assays (fig 5). a direct way of studying receptors. not til about 1970 could they get radioligands - labelled ligands that interact with the receptors. what they do is isolate a tissue homogenate that has the receptor in it. they incubate it with radioactive ligand for some time until equilibrium is reached b/w free and bound drug. then they separate unbound drug from the receptor-drug complex (by filtering the material), and they end up with the complexes, and they count based on radioactivity of the material. that's a good way of directly measuring receptor drug interaction. it's called "grind and bind" method :) another method is receptor autoradiography. this is very similar to the radioligand binding, but instead of tissue homogenate they take a whole tissue slice and mount tissue section on a slide, dip it into radiolabelled ligand, let it equilibrate, rinse off unbound ligand, and put it into a radiograph cassette with a film - the bound receptor will expose the film, so you can see where the receptor is in the tissue. slide: slice of rat brain that was used in receptor autoradiography. wanted to find the AT receptors in the rat brain. the dark areas are where the receptors are - the paraventricular nuclei.this method gives you anatomical localization of receptor on the tissue, unlike the grind and bind method. it's more difficult but worth it if you need the anatomical localization of your receptor. you can also quantitate by the density of the dark area on film being proportional to amount of receptor. criteria for being a receptor: 1. saturability - this means that for a tissue or cell, there is a finite number of receptor sites, so when you give a drug, if you give a little, you get a small effect, and increasing dose increases effect, but at some point effect will plateau because you have saturated the receptors. 2. specificity - this means that only those cells which have the receptor will respond when that receptor is activated. This was noticed by langley in 1905, leading to the hypothesis of receptors. only those cells with the receptor will respond to the ligand. Also, the receptor must have high affinity for the ligand. There should be a correlation between amount of ligand and level of biological response. Say you take a cell and sit it in isotonic saline buffer, and then throw in 10x the normal amount of NaCl into the solution. The cell will have a response, but not due to a "salt receptor" or anything - just because the osmotic pressure creates nonspecific effects. Specificity implies specific effects of something within a physiological range. 3. reversibility - when we apply ligand to the receptor, it binds the receptor, and nothing happens to the ligand - it isn't altered by the receptor - it can pop back off, unaltered. if ligand is altered, than "receptor" is an enzyme, not a receptor. 4. bifunctionality - recognition/binding and initiation of cellular response keep these points in mind as we discuss the ways in which we can analyze the receptors. Types of analysis: 1. saturation analysis - you vary the concentration of radioligand and measure the formation of receptor-ligand complexes. so you measure amount of receptor binding with increasing amounts of radioligand being added. see fig 7. you incubate a known quantity of tissue with increasing concentrations of radioligand - see three curves in the diagram. the total curve is what you get when you just add radioligand, flush off unbound ligand, and look at total radioactivity - total binding. when you do this kind of assay, realize that the ligand can bind other things aside from the receptor - it has a certain stickiness that is somewhat resistant to washing. it can stick to other membrane proteins, or to the slide, or whatever. you want to correct for that. so they incubate with unlabelled ligand at every concentration of radioactive ligand - flooding receptors with unlabelled ligand so that they aren't occupied with radioactive ligand - and get the amount of nonspecific binding, which you then subtract from the total binding to get the specific binding. note the curve is a rectangular hyperbola which obeys the law of mass action. when ligand binds receptor, it isn't static - it binds, pops off, binds, pops off. note also that at some point on the specific curve it flattens out, but that the nonspecific binding curve never flattens. from this type of curve we can get some numbers to make an index of how tightly it binds the ligand. when you reach the plateau amount, you know the total amount of receptors for that tissue. you extrapolate that to the y axis to get the V max - maximal amount of receptors in the tissue. the concentration at which 50% of receptors are bound is the Kd, the affinity constant of the drug for the receptor. comparing Kds gives an indication of how tightly one drug binds a receptor compared to another. the lower the Kd, the higher the affinity. this saturation analysis is often transformed into a linear plot called a scatchard plot, because it is hard to get an exact Kd off a hyperbolic graph. So, they would make it linear so they could just use a ruler. nowadays, they can use computers to figure out the Kd from the rectangular hyperbola without using the Scatchard analysis. but, it's still useful to make the Scatchard analysis because you don't need to use such a wide range of ligand concentrations. ----break---- 10-11 to continue... remember those receptor criteria as we go through the types of analysis. The saturation analysis gives us some hard numbers - Kd, Vmax. next type of analysis - Competition/Inhibition experiments. rather than vary the concentration of radioactive ligand, you vary the concentration of an unlabelled competitor. fig 8 in handout. the level of specific binding is plotted against the amount of nonlabelled competitor. At low concentrations of competitor, all the receptor is bound - but as you increase the concentration of competitor, you displace labelled ligand with unlabelled competitor. analyzing a wide range of competing nonradioactive ligands, you see that they displace at different concentrations. some displace earlier at lower concentrations than others. this can give us an index of comparison for relative affinities of compounds for a receptor. IC50 - inhibitory concentration 50 - concentration of nonradioactive ligand which leads to 50% specific binding. each compound you test has an IC50 so you can kind of get a rank/index of potency this way. This is a fairly complex chart in fig 8. he also showed a slide that has fewer lines on it - we see that one of the competitors doesn't bind the receptor at all so curve stays at 100, then we see a couple of other curves - note that two are nice and sigmoidal, showing monophasic displacement of the labelled ligand. another one is biphasic - what does that mean? a biphasic displacement indicates that possibly there is more than one subtype - here, there is a high affinity and low affinity binding site for this particular compound, which isn't present for the other compounds. the first plateau indicates saturation of high affinity subtype, then it starts binding the low affinity subtypes. so far we're only discussing ligand-receptor binding - remember this is a dynamic state, and at equilibrium, ligand is constantly attatching and detaching. dose-response rate - what happens to cell after binding of ligand to receptor? fig 9 in handout - plots response vs dose and it shows a rectangular hyperbola, because it obeys the law of mass action. now, when we talk about response, you don't have to limit yourself to molecular changes in the cell. it could be a tissue or organism response, as long as you can measure it and it is quantifiable, you can define your response however you like. You can also plot response as a percent of maximal vs log of dose - this gives a nice sigmoidal shape. this semilog form is the way dose response curves are usually plotted, because you can use a wider range of drug. he's showing us a slide where he's measuring amt of cAMP in response to varying concentrations of different drugs. we see sigmoidal curves that reach plateaus at high concentrations - saturability - also that some curves do not overlap, some are shifted right or left - a drug which creates a response at a higher dose is not as potent. potency is related to receptor affinity. using an antagonist produces a flat curve because no cellular effects occur. you only see response with an agonist. AT I produces a response, but not as much a response as the other drugs on this chart. so realize agonists can have partial effects - AT I is a partial agonist for this receptor. Its intrinsic efficacy is lower than that of a full agonist. a full agonist produces maximal cellular response, and is given an intrinsic efficacy of 1. an antagonist is given an intrinsic efficacy of 0, and partial agonists are between 0 and 1. cartoon diagram: shows an agonist and an antagonist binding to a receptor. the antagonist binds the receptor at certain places but doesn't interact with a key area of the receptor - just covers it up. the agonist interacts with the key area and produces a response as well as binding to the other parts of the receptor. a partial agonist binds to only part of the key sites. so when you consider drug effects you have to include knowledge of affinity and intrinsic efficacy - if it is a full or partial agonist. there is a whole series of exceptions that will depart from the simple things we've been discussing....see fig 10 in handout. we see a binding curve and with increasing dose of the drug the curve shows you the occupancy of the receptors. Kd is plotted for us. A shows us that fewer receptors are being occupied but we're still getting an appreciable response - because there are extra receptors - maximal effect occurs when only a small percent of receptors are occupied. this creates a lot of sensitivity to a particular compound. so body can react to very tiny hormone signals. this shifts the effect curve to the left, as in curve A. or, you can shift effect curve to the right as in curve B, where effect lags behind percent of receptors bound, because you have to have many receptors occupied to produce the effect. note also that sometimes when you give a drug chronically, the efficacy of the drug may become diminished. so you start giving more drug to try to create the therapeutic effect - until you reach toxic level. this is a frequent problem. this phenomenon is called receptor desensitization and downregulation - see fig 11. this is the beta-adrenergic receptor treated with an agonist, isoproterenol. you have the receptor present, you add the agonist, it activates it, g protein response occurs, etc - but once the g protein is activated, it isn't on forever - it acts for a certain time. eventually it has to reassociate to the receptor to be reactivated. but with this agonist, the same cellular actions that initiate the cellular response will also cause desensitizaton of the receptors. a compound forms that prevents the receptor from recoupling to the G protein, by keeping it phosphorylated. so the receptor can't initiate more cellular responses. it can bind agonist but not do anything. so it is a desensitized receptor. a separate process coupled to this is receptor downregulation - cells actually remove the receptor from the cell membrane. this can occur after about 8 hrs of tx with isoproterenol. this isn't permanent. you remove the agonist, and the cell can put the receptors back into the membrane or can remove the phosphorylation to resensitize it. this is important. you may have to give a drug holiday/drug vacation from a particular drug, or give a drug that operates on a different receptor system, to give the affected receptors a chance to be resensitized and returned to the membranes. the opposite effect is receptor supersensitivity. this is where extra receptors are produced as a response to a drug. when you have presynaptic and postsynaptic membranes, as at the neuromuscular junction, and you remove the presynaptic input due to injury, as you know the postsynaptic membrane will get covered with a proliferation of receptors, so that even the smallest amount of agonist will be more likely to bind receptor. the final point is the effect of antagonists on the dose/response curve. look at fig 12. this graphs perception of pain vs dose of morphine. we're measuring the elevation of pain threshold. as you give morphine, you see an increase in the pain threshold. when subject is pretreated with a competitive antagonist for the morphine receptor, we see that you have to give higher doses of morphine to get the same effects. you can, however, still reach full effect, but the curve is shifted to the right. this is due to the dynamic situation of ligand-receptor binding. when using a noncompetitive antagonist - this is a drug that may destroy a receptor, or act at another site somewhere - you can not overcome the effects of the antagonist, because it's acting elsewhere or removing receptors. the curve is shifted to the right AND has an early plateau. ---break--- 1-2 Davies - Drug Biotransformation How do animals cope with poisonous substances (drugs)? If you put a drug into an animal and do nothing to the drug, the drug will remain in the animal forever. As Dr. Yee explained, it will just pop on and off the receptors and will maintain its activity. So, there must be some way to terminate the activity of drugs (and endogenous chemicals). There are three ways. 1. excretion 2. dilution in the body via distribution, protein binding, fat binding 3. chemical transformation of the drug via biotransformation drug metabolism - total fate of the drug in the body biotransformation - chemical transformation of a drug in an organism, usually by enzyme catalyzed reactions. How did biotransformation evolve? slide: volcano erupting. this is a pollutant. Even oxygen is a pollutant - bodies need to adapt to these pollutants. Slide of penguins evolving :) Animals have developed chemical systems to detoxify foreign (and deactivate endogenous) substances. Plants lack excretory systems - they neither urinate nor defecate - but they take toxic substances and put them into vacuoles. They put them away so they aren't part of the systemic cytosol of the cell. These toxic substances in vacuoles may potentially be eaten by animals. So, animals may eat a tobacco plant and get a dose of nicotine. these plant toxins are likely to be fat soluble and easy to absorb via GI tract, and hard to get rid of. So animals need a way to cope with these toxins. quick history- fish have large gill surfaces and a lot of water runs over gills. they can excrete toxins through gills into what for fish is a relatively infinite volume of water, because there is such a high flow of water over the gills. most fat soluble toxic substances aren't too problematic for fish. but water soluble toxins are big problems for fish. land animals (slide of kangaroo) had to evolve to conserve water. the mammalian kidney makes very concentrated urine. If the urine is highly concentrated, substances in the urine are highly concentrated, so fat soluble toxins are concentrated in the urine and then easily reabsorbed back into systemic circulation. So, for mammals to cope with toxins, they have to convert them into water soluble substances - more polar. So, they evolved a group of enzymes which will biotransform toxins, drugs, endogenous substances, into polar, water soluble substances that are easily excreted in the urine. I. Significance: 1. high water solubility permits efficient excretion of a drug in a limited volume of water in the urine or bile. Therefore, the common feature is that enzymatic actions yield products that are more polar (water soluble) than their precursors. (see above) 2. Animals adapted enzymes that normally biotransform endogenous substances: steroids, bile acids, thyroid hormone, prostanoids, fatty acids Now, animals had to evolve systems to deal with toxins. How did this come about? most tissues in the body have enzymes that biotransform endogenous substances. all tissues but RBCs and skeletal muscle will do this biotransformation to synthesize necessary compounds. The adrenal will transform steroids, the liver will biotransform bile acids, etc. natural biotransformation/synthesis is normal in all mammals - but *see below* 3. These enzymes used on endogenous substances can act on exogenous substances if these exogenous substances have enough molecular resemblance to the natural substrates (endogenous substances). 4. consequences of biotransformation: A. Activation: inactive compound transformed into an active compound. this is NOT the most common reaction. You can take Parathion and transform it into paraoxon, or L-dopa to dopamine, or benzpyrenes to epoxide, CCl4 to free radicals, and aromatic amines to hydroxylamines which cause methemoglobin to form, and acetaminophen to a toxic intermediate. acetaminophen is activated by cytochrome P450 to a reactive intermediate which can do cell damage. B. No change / Maintenance of activity - diazepam is biotransformed to oxazepam which has the same activity C. Inactivation - phenobarbital transforms to hydroxypentobarbital, which has no biological activity. this is the most common reaction (inactivation). 5. sites of biotransformation. Liver is the most important organ of biotransformation, although it can also occur in lung, kidney, intestine, skin, and gonads. Liver is really the main player. The smooth ER of the liver is the most important site in the most important organ. The enzymes here are membrane bound. If you destroy the membrane, you eliminate enzyme activity, can't metabolize the drug. the membranes are arranged in a chain sitting in the membrane. II drug transformation reactions - divided into two phases, I and II. 1. phase 1 occurs in microsomal enzymes of smooth ER (also mitochondrial, cytosolic, and other sites, but mostly SER of liver). They change the molecule, forming functional groups, making the compound more reactive. They can cause oxidation, reduction, hydrolysis; activation, inactivation, or no change to the molecule - mostly inactivation. The product, being reactive, may then undergo further (phase II) reactions. 2. phase II reactions are not limited to microsomal enzymes of SER - also mitochondrial or cytosolic enzymes. these are synthetic reactions - you are going to add something to the phase I product. conjugation or other synthetic reaction. almost always causes inactivation. see diagram on p 2 of handout PHASE 1 reactions: -catalyzed by C-P-450 -basic process is hydroxylation (it's an oxidation) - adds hydroxyl group. - NADH + NADPH + RH2 + O2 --> NAD+ + NADP+ + RHOH + H2O - NADH and NADPH supply H+ and electrons -the H+ receptor is oxygen (one oxygen goes to the drug, one to the receptor, so it's a monooxygenase because one oxygen goes to the drug, or a mixed function oxidase because one goes to drug and one to water, or a DME - drug metabolizing enzyme) -analagous to mitochondrial electron transport. reaction steps: 1. drug binds to Fe+++ on enzyme 2. electron transferred to Fe+++ (ferric) to make Fe++ (ferrous) 3. enzyme-Fe++ complex binds O2 4. another electron and H+ is added from NADH --> FeOOH (activated oxygen moiety) 5. H2O is released --> (FeO)+++ (more highly activated oxygen moiety) 6. (FeO)+++ directly oxidizes the drug back to ferric state --> hydroxylated drug and Fe+++ produced 7. Fe+++ now binds a new drug molecule see p 3 of handout. The details are not important. Know that the drug is oxidized and a hydroxyl group is added to the drug. Cytochrome P450 - a family of enzymes, of heme proteins - there are over 20 of them, probably over 40. each one can metabolize different substrates. some can metabolize only one or two compounds, others 30 or 40 compounds. Depending on the substrate, different reactions occur. The main thing is at least as an intermediate compound,a hydroxyl group is always added at some point. that's the basic reaction. the amount/# of molecules of a particular P450 enzyme, and their activity, will increase in response to inducers. The enzymes that are in the membrane; the inducers are all highly fat soluble substances that stay in the membrane, and hundreds of drugs can induce enzyme activity. the inducer may be a substrate - and if it is, if the enzyme is acting on a drug which becomes an inducer, then it will increase the degradation of itself, leading to tolerance of the drug (before we discussed the phosphorylation of a receptor leading to drug tolerance, this is another way of causing drug tolerance - making the inducer a substrate). many but not all substrates are inducers. some inducers affect the metabolism of many drugs, eg phenobarbitol. if you give a drug metabolized by p450 and also give phenobarb, the metabolism of the second drug is increased. usual example is phenobarbital and coumarin - you have to give more coumarin b/c the metabolic degradation of coumarin is increased by the presence of phenobarb - and if you stop the phenobarb you have to decrease coumarin dose - withdrawal of an inducer should cause you to change the dose of a drug. environmental agents may be strong inducers - PCBs, dioxin, chlorinated hydrocarbons, polycyclic hydrocarbons, barbecues, tobacco smoke (polycyclic hydrocarbons). Rate limiting factors: 1. hepatic blood flow - delivery of drug and O2. if you change hepatic blood flow you change these parameters. So, if CO changes, then metabolism will change. 2. amount of NADPH-C-P450 reductase can be rate limiting step (transfers electrons from NADPH to P450 3. amount of NADPH and NADH - usually ample but decreased with starvation so starving animal can't metabolize drugs as well 4. competition for enzyme binding sites - CO, cimetidine, erythromycin, ketoconazole. 5. noncompetitive inhibitors- halogenated hydrocarbons, heavy metals (affect heme synthesis), inhibition of protein synthesis (enzyme synthesis), inhibition of P450 synthesis - people are working on that to try to stop some tumor growth by preventing biotransformation of tumor inducers. Other phase I reactions: 1. sites: mitochondrial, microsomal, cytoplasmic, lysosomal, plasma 2. hydrolysis: split esters (acid + alcohol), acetylcholinesterase (at neuromuscular junctions), butyrylcholinesterase (a plasma cholinesterase, breaks down local anesthetics eg procaine). 3. Oxidases - alcohol dehydrogenase (cytosol) - breaks down alcohol into acetaldehyde (which gives you a headache) which is then further metabolized in mitochondria by acetaldehyde dehydrogenase, into acetate. phase II reactions: 1. synthetic - most important is glucuronic acid - adds glucuronic acid, sulfate, acetate, glycine, glutathione, etc. 2. makes compound more water soluble 3. catalyzed by cytoplasmic, mitochondrial or microsomal enzymes 4. glucoronic acid is most important conjugate - not free acid - won't couple - need uridine diphosphoglucuronic acid and a transferring enzyme which is deficient in cats and newborns. newborns don't handle exogenous (and some endogenous) substances as well as adults. when newborn changes from fetal Hb to adult Hb, the bilirubin that's released isn't metabolized as quickly, it gets bound to plasma proteins and produces jaundice. if you give something that displaces bilirubin off the plasma proteins, bilirubin can precipitate in tissues, esp in brain and cause kernicterus of brain nuclei. 5. reactant is transferred to an electron rich atom on the substrate. factors affecting biotransformation: 1. age - low activity in young and old 2. nutrition: affects NADPH and NADH levels, can have deficiency of enzyme cofactors, conjugation reactants, can cause decreased plasma proteins 3. disease: renal - increased plasma proteins; inflammatory: change in plasma proteins, alpha1-acid glycoprotein; hepatic: decreased plasma proteins, diminished biotransformation, altered hepatic blood flow. see diagram on p 8 - note that regenerating liver shows increased biotransforming activity 4. inducing agents - will increase amount of enzyme available, increasing biotransformation 5. inhibitory agents - lead/other heavy metals - interfere with heme synthesis. may be used therapeutically - MAO inhibitors, tricyclic antidepressants. they may act by altering circulation to the liver or competing for enzyme binding sites (ethanol and ethylene glycol - use same enzyme binding site); competing for endogenous substrates or conjugation substrates 6. genetic differences - species differences/enzyme deficiencies. eg, cats have specific enzyme deficiency, dogs are poor acetylators and don't handle sulfonamides as well. see table in handout. if you give hexabarbitol in equivalent dose to various animals, mice will sleep 12 minutes, rabbits 49, dogs over 300 - due to differences in oxidation by liver enzymes. ---break---- 2-3 Dr Davies remarks that he has never before lectured on "response variation to drugs" and that there are only a few concepts, and tons of details. the stuff left after removing the examples is the substance of this lecture - it's about three lines of text. So focus on this. The problem with the details is one, they are details, and two, we haven't learned about the drugs yet, so we don't know what they do. Dr. Robinson already told us about age was important, and then Dr. Davies mentioned it again as important for biotransformation. Robinson also talked about changes in pH due to disease, or other drugs, and how it can affect distribution, concentration in different compartments, and how you can use it as a mechanism for antidotal treatment. example: suppose a dog got into your aspirin, and has aspirin toxicity. this leads to CNS signs, among other things. so your dog is hypermetabolic, and you want to treat it and get rid of the aspirin? well, it is a weak acid. one way to get rid of a weak acid is to put it out in the urine. You can alkalinize the urine, so that the weak acid will donate a proton and become ionized and therefore effectively trapped in the urine - unable to cross the membrane and reenter the body. How do you do this? there are a few ways. one thing you can do is give the animal NaHCO3 to raise pH of urine, or to use a diuretic which inhibits carbonic anhydrase, which will also raise the pH of the urine. The aspirin is also in the brain. If you want to get the aspirin out of the brain, you should probably also make the blood more basic, right? You want to trap it in the blood. So, it's probably better to give the bicarbonate which will also make the blood more alkaline. Carbonic anhydrase inhibitor acidifies the blood - you wouldn't want to do that. route of administration affects concentration of drug, at least acutely, as will concentration given (dose), and this will affect the response. as drug concentration changes, response changes. if you have receptors that are responsive to the drug, and the receptors have different affinities, as you raise the concentration you will bring in new receptors of differing affinities. if you give morphine to a dog, you should give it subQ because you don't want to get the concentration too high - if you give it IV, it may bite your face off, because it will get excited, because you activate a different subset of receptors. If you give morphine to cats, they can also get excited, unless you stay with low concentrations to avoid the other subset of receptors. most of the response variation we're going to deal with for drug interactions are relatively minor. the horror stories are when it isn't minor, but most interactions are minor. a lot of response variation is theoretical - hard to measure. but sometimes can be strong or even fatal, so you have to know what's going on here. sources of response variability: types of response variability: 1. pharmacokinetic - different concentrations of the drug at the site of action, despite the dose being the same - eg, give same dose to two animals, get two different responses due to two different concentrations of drug at site of action. this is most important. due to variations in absorption, distribution, metabolism, and excretion. 2. pharmacodynamic - this means that you can get differing responses to the same concentration of a drug at the site of action. Depends on which receptor types are present, how many receptors are present. some receptor types change with maturity - some are absent at birth and show up later. receptors may be upregulated or downregulated, causing pharmacodynamic response variability. there may be biochemical changes in the cell - uncoupling of second messenger systems from receptors due to phosphorylation of receptor, as discussed with isoproterenol. also physiological responses - interacting organ systems - depending which organ system is activated and how it responds, response can vary. eg, blood pressure can be controlled in different ways, and the response can vary - eg, if heart contractility isn't good, response to drug that increases contractility may be poorer than in healthy animal. before giving the drug you should be aware of factors which may change the response: 1. genetic factors (breed/species) - there are thousands. 2. effects of age - can affect physiological function - glomerular filtration and CO decrease with age, maximal breathing capacity decreases with age...so response to drugs will change with age. reduced CO will limit distribution...reduced filtration will limit excretion. The halflife of diazepam increases drastically in old people! see handout p 1, diagram under effects of age. - pharmacokinetic changes - absorption changes, and distribution changes. acid secretion is reduced in the aged, reducing absorption, as is splanchnic blood flow. there is loss of muscle mass and increased fat, so water soluble drugs have a lower volume of distribution. things bound to muscle won't be as bound. fat soluble drugs will have increased volume of distribution. plasma proteins are reduced in aged patients too, changing concentrations of drugs bound to plasma proteins. Neonates also have reduced plasma binding. metabolism of drugs - there is decreased liver enzyme activity in aged animal, reduced hepatic blood flow, reduced liver mass. infants also have reduced hepatic enzyme activity. renal excretion - low blood flow and glomerular filtration in aged patient, also low in neonates. so excretion is reduced. these are all pharmacokinetic changes that occur with age change. -pharmacodynamic changes - receptor numbers are low in neonates, and neonates will have different receptor sybtypes and altered affinities. older animals also have altered receptor sensitivity, altered coupling to receptors, altered physiological responses in both neonates and aged patients - eg ability to vasoconstrict or whatever. 3. history of drug use (drug induced loss of response) - we talked before about tolerance - seen after chronic drug administration. takes a long time to develop - days, weeks, etc.tachyphylaxis is an acute loss of response to drug - within minutes. this can take place through changes in receptor conformation, loss of receptors, exhaustion of mediators (eg, amphetamines deplete NT, so there is no response to more drug), increased metabolic degradation of drug (upregulation of P450 enzymes), physiologic adaptation - reflex or biochemical compensation - eg if you give diuretic that inhibits carbonic anhydrase, it will cause acidosis, so there will be reduced bicarb in urine - less is secreted, and less of a response to more drug. 4. health status - effects of disease or altered physiologic states (pregnancy) there are thousands of reasons why this affects response to drugs. we won't discuss this. 5. drug interactions - what other drugs animal is taking many and varied. thousands to remember, impossible to remember them all. for those drugs that you use often and in acute situations, know the interactions. the rest you have to look up in the PDR or VPB. most of the drugs you use are "dirty" drugs that affect many different receptors. some of the receptors they affect are the ones you want them to affect and others are not. consider three drugs: diphenhydramine, chlorpromazine, and procaine. they all share a common side chain and have a lot of common effects: local anesthetic, antihistaminic, antiarrhythmic, anticholinergic. if you give them together, the shared effects are increased. if you give drugs in combination, you will probably have overlapping affects. note that also rarely is only one drug used - combination drug therapy has lots of advantages, and most often you have more than one drug on board - eg, people often start out with nicotine and alcohol already on board! as the number of drugs increases, the possibility of interaction incraeses. most interactions are mild, some are severe. but it's important to know what drugs the animal is taking. classification of drug interactions: can be classed by consequences - beneficial or adverse can be classed by site of interaction - external (in vitro), drugs that can't be mixed in the same solution without producing precipitate, or heat, or something; or internal - in the body mechanisms of interactions: physiological - two drugs act at different sites to alter function - can affect heart and also arterioles to affect blood pressure pharmacodynamic - one drug changes the effect of another drug -usually predictable if you know the pharmacology. many interactions will be discussed in future lectures. drugs can interact indirectly - can change pH, which then affects activity of other drugs. can affect the ion environment of cells -so if you give a certain diuretic, you lose K+, which will affect digitalis and its activity on the heart. pharmacokinetic interactions: one drug changes the concentration of another drug. these are often not predictable. if you change gastric emptying rate, or pH of GI lumen, you affect absorption of other drugs. if you damage the mucosa with an NSAID, you affect rate of absorption of other drugs. you can change the distribution, the CO, the blood pH, etc. alterations in active transport also change distribution. Guanethidine, used to lower BP. if youput desipramine into the animal at the same time, it affects reuptake of norepi, which raises the BP, so these drugs interact by changing the affects of a third compound. you can change protein binding by giving a competitor for the binding site- you can give a displacing drug that will change the protein binding of bilirubin, for example. if you have a protein bound drug, and if you give a displacing drug, you change the amount of free drug by displacing it from its protein binding site - so you are increasing the drug activity, raising potential for toxicity. this used to be considered very important but now we know that if it is going to be a problem, it will cause an acute transient toxicity, then resolved by redistribution of displaced drug throughout its volume of distribution - reducing concentration in plasma - and then going on to have increased elimination. so this limits any toxicity that may result. You end up with about the same concentration of unbound drug you had before, but less total drug - same amount of unbound drug. if elimination is impaired, toxicity can result. neonates have impaired elimination. patients with cirrhosis or renal disease may wind up in trouble from displacement also. another drug interaction - metabolism/biotransformation - one drug can induce enzymes that alter metabolism of another drug, like phenobarb and coumarin. this is especially important if drugs undergo extensive first pass elimination. So, if you give phenobarbitol to an animal on coumarin therapy, this increases metabolism of coumarin, so you have to increase coumarin dose. if you withdraw the phenobarb, you have to decrease the coumarin dose to prevent bleeding. excretion - one drug can alter excretion of another drug. glomerular filtration can be altered by altering plasma protein binding; tubular secretion can be altered by having a drug compete for an enzyme site (using a drug to prevent penicillin from being secreted) adverse reactions: these are serious but relatively infrequent toxicities, sometimes causing death, which are observed at the usual therapeutic dose. sometimes called idiosyncracies. they are not detected in preclinical studies because they are infrequent, or the proper population wasn't studied. malignant hyperthermia - halothane aplastic anemia - chloramphenicol adverse reactions may be dose related, or not dose related. in general, drugs are tested on healthy animals or for a particular disease - but drugs for one disease aren't tested on people with another disease, and the adverse reaction can be due to presence of another disease - or due to genetic variation, or something. if it's not dose related - could be allergy, or unknown - then you can't use that drug in that patient at all. adverse reactions are more common in older animals and neonates, females, and when multiple drugs are given. it's important to do postmarket monitoring - report adverse rxns to the CVM/FDA. this is the only way to find some of the problems with some drugs. ---end---