---start pharm.lec.01.07.98----- pharm 1/7/98 RO Davies 9-10 scheduled lectures: continued general principle review; drug translocation we were discussing clearance (CL), a measure of the ability of an organ or system to remove substances from the blood or the body. the concept is useful if you're considering giving drugs, you want to give an amount equal to the amount able to be eliminated so you do not accumulate too much drug in the body. amount in should equal amount out, and amount out is determined by clearance. remember amount = concentration x volume. recall from physiology we discussed delivery of oxygen to an organ or organism in terms of the amount delivered equalling the amount in minus the amount out. so blood comes in, drops off the oxygen, and comes out, containing less oxygen. the amount of missing oxygen is the amount that was delivered to the organism. if you had a number of people in a bank with $100 each, and when they left the bank each had $90, and at the end of the day the bank had $1000, how many people were there? that's simple. but if you replace the people with blood and the money with concentration of oxygen in arterial and venous blood, it is more difficult for us, even though the concept is the same. so consider, tissue uptake or tissue elimination from blood is equal to amount in minus amount out, and amount in is the blood flow times the concentration in arterial blood and amount out is blood flow times concentration in venous blood. also, we're considering for flow that all the blood is participating equally, just as we're considering that each person dropped off the same amount of money. that may not always be the case. what is important is the blood flowing through the organ of elimination. if it's the liver, you have to look at liver blood flow in minus liver blood flow out, or whatever. Dr Bovee discussed the concept of clearance before - he was considering that if you have some urine with stuff in it, you need to know how it got there, and the only way it did that is if the kidney took it from the blood and put it into the urine. So consider clearance for the kidney - this is: amount in blood = amount in urine volume in blood x concentration in blood = volume in urine x concentration in urine so... volume in blood = volume in urine x concentration in urine ----------------------------------- concentration in blood consider the people problem again. how many people have to be cleared of money to get $1000 into the bank? ten people have to be cleared of all their money. but people do not participate equally - some are cleared of some money, some are cleared of all, some of none. clearance is the amount eliminated with respect to the concentration - ability to get rid of a substance, that's what clearance means. dosage rate should equal elimination rate. again, clearance is a volume of blood that must be cleared to account for elimination. so dosage rate should equal elimination rate. elimination rate = clearance x concentration = dosage rate how do you determine the concentration of a drug? it's set. that's how you buy it. so that's a known value, set by the veterinarian. so to know what the dose is, you have to know the clearance, too. if clearance changes, dosage rate must change also. what will change clearance? clearance depends on blood flow, and if blood flow changes, CL will change. If CO changes, clearance will change. cardiac patients will have altered clearance. Intrinsic function of the kidney will also affect clearance. the kidney is an organ of elimination. damaged renal tubules can't eliminate drug as well. clearance will change so dosage must change. damaged liver will not eliminate certain drugs well - clearance will change, so dosage must change. clearance changes with age. neonatal animals have poor drug metabolizing systems - so doses must be different. glomerular filtration in the aged is reduced, so their clearance is changed, so dosage must change. a lot of old people get tranquilizers at inappropriately high doses because they have reduced clearance and the drug stays in their bodies too long. so always consider this. elimination could imply metabolism - liver can metabolize drug, convert it, oxidize it, add a conjugate, and eliminate it - but at this point it isn't the drug any more. Or, something could just be eliminated via glomerular filtration. different drugs are eliminated in different ways. Garlic is eliminated via respiration - you breathe it out. you give a drug. you raise the blood level. it diffuses into liver. it is metabolized. as this occurs, blood level drops. this takes time. you want to keep the concentration at an adequate level, constantly. this is really impossible to do except in an ICU or laboratory. practically, we keep it in a range above the therapeutic level and below the toxic level, but the concentration will yo-yo between those levels. moving on... another thing to consider is extraction ratio: amount in - amount out = extraction Q (arterial concentration - venous concentration) = extraction [note to self -> flow = Q] so, the extraction ratio is what is extracted divided by the original concentration: Extraction ratio = [art. conc. - venous conc.]/art. conc this determins how fast drug gets into tissue - a high ratio means elimination depends on blood flow, not intrinsic function of the organ, because that function is very high, it's extracting. intrinsic function of an organ with a higher extraction ratio isn't important b/c it can take everything up. if the extraction ratio is low, if the organ can only extract a little of the drug that is brought to it, then changing organ function will be very important, so if you can induce more enzymes in the liver you can increase elimination, because elimination depends on function - more enzymes increases elimination. binding to plasma proteins is important, because the organ can't handle bound drug, it can only handle free drug. so the point to consider is what percent of the drug is taken up. the last pharmacokinetic term to discuss is half-life. this is defined as the time needed to reduce the concentration of a drug to one half of its starting value. If you have a drug that is easily cleared, it will have a short half-life. half life is proportional to 1/CL. the other factor in half-life is you can only clear a drug that is in the blood. if it isn't in the blood you can't eliminate it. so drugs with a high volume of distribution elsewhere, outside the blood, bound to muscle proteins or dna or whatever - can't be cleared. so half-life is proportional to volume of distribution, and half-life is proportional to volume of distribution divided by clearance. If drug is bound in fat, muscle, anywhere but blood - it can't be cleared. Environmental toxins are often stored in fat and not cleared unless the animal is starving and fat is mobilized. so CL changes with heart dz, liver dz, kidney dz. Vd changes with amount of fat, protein binding tissue, muscle mass, cell permeability so all those things affect drug half-life as well. note: many drugs are not effective in the brain unless you have meningitis - inflammation changes permeability. law of mass action: most drugs act at low concentrations and have very specific effects. they have specific effects because they bind to tissue receptors on cell membranes. Receptors determine the quantitative relationship b/w concentration and effect. If you have a drug (most drugs), the drug + the receptor will bind to form a complex DR, and that will produce a response. The law of mass action says that the rate of mass action is proportional to the concentration of the reactants. in order to react, chemicals have to meet and interact, and the amount of interaction between chemicals depends on their concentrations - just as the interaction between people in a room depends on the number of people in the room. so if you want to form a DR, then the rate of formation will be as follows: Form DR --> k1[D][R] dissociate DR --> k2[DR] at equilibrium, the formation of DR equals the dissociation of DR k1[D][R] <===> k2[DR] so... [D][R] k2 ------ = ---- = Kd (the equilibrium dissociation constant) [DR] k1 Kd is the concentration of drug required for 50% of the receptors to form complexes. This is in the handout somewhere. so if you look at the concentration of drug required to produce an effect, you find that you increase the effect with increasing drug concentration until you saturate the receptors, at which case there is no increase in effect. so at low concentrations of drug, effect is proportional to concentration, and then as you increase it at higher concentrations, the effect levels off. when 50% of the receptors are saturated, you're at the Kd. this is a measure of affinity of drug for the receptor. If you need a high concentration of drug to get 50% of receptors to complex, you have a low affinity. in physio we discussed p50 as a measure of affinity of hemoglobin for oxygen. now we're measuring affinity of drug for receptor. It doesn't really have anything to do with how effective a drug is - it can have low affinity and be very effective - like many NTs, which have very low affinity and high dissociative rates. Henderson-hasselbach: this is also in the handout, and relates to the law of mass action. 1. ionization of water H2O <==> H+ + OH- Ka = [H+][OH-]/[H2O] the concentration of water is very high compared to the concentration of dissociated products, so the degree of dissociation has little effect on the concentration of water itself, so therefore we talk about the K of water, Kw, as being the concentration of [H+][OH-], which is 10^-14. because that is a large number, someone decided that pH would be -log [OH-] 2. Acid = proton donor H2CO3 <--> H+ + HCO3- bases = proton acceptors NH4+ <===> H+ + NH3 so ammonia is a base, and bicarbonate is a conjugate base. many drugs are weak acids or weak bases. most weak bases are ammonia compounds. acidity drives both of the above equations to the left - adding hydrogen ion drives equation to the left. acidity makes acid nonionized and makes bases ionized. 3. Henderson said: H2CO3 <==> H+ + HCO3- Ka <==> [H+][HCO3-]/[H2CO3] 4. Hasselbach said: log Ka = log [H+][HCO3-]/[H2CO3] log Ka = log [H+] + log [HCO3-]/[H2CO3] log[ H+] = log Ka - log [HCO3-]/[H2CO3] -log [H+] = -log Ka + log [HCO3-/[H2CO3] pH pK so pH = pK + log [HCO3-]/[H2CO3] this is all in the HAndout. we will see how pH affects ionization of acids and bases and how easily things cross membranes. the important thing to remember is that acidification drives acids to nonionized form, and drives bases to ionized form. ---break--- 10-11 In general, the intensity of drug action and the duration of drug action is directly related to concentration at the site of action. This is *in general*. We want a) an adequate drug concentration, and b) continuously maintained concentration. That's what you want. It isn't what you usually get. This depends upon: 1. route of administration (IV, PO, IM, SQ, etc) 2. translocation of drug molecules (moving them around) 3. rate of biotransformation (lecture 7)(what organs do to drug chemically) 4. rate of elimination (lec 3, 4) movement of drugs in the body - only two main ways to accomplish this. one is bulk flow, by convection, in the blood; and two is by diffusion. some are actively transported, but the main ways are bulk flow and diffusion. bulk flow is in the circulation, moves drugs long distances. the chemical nature of the drug has little effect. the blood doesn't care if it is fat soluble or not, as long as it is in the blood it will be transported. CO determines speed with which drug is distributed through body. regional blood flow also plays a role. The other way that drugs are moved is by diffusion. Diffusion takes place over short distances. diffusional characteristics of drugs vary greatly - important point! especially the ability of drugs to cross cell membranes/lipid barriers. they must dissolve in membrane, fill up membrane, then spill over to other side of membrane. cell membranes separate the various aqueous compartments. they are nonaqueous lipid barriers. capillary endothelium separates intravascular from interstitial. cell membranes separate extracellular from intracellular. cell membranes separate extracellular from transcellular compartment. aqueous diffusion - this delivers drugs to and from cell membranes - in the stomach, drugs must diffuse in gastric fluid to the wall of the epithelium. that's governed by Fick's law of diffusion which is in the handout. diffusion of a gas, or flow of a drug in our case, depends on the area, which is very important for drug movement across GI epi, it depends on the permeability coefficient, and on the difference in concentration. it is also inversely proportional to the thickness. the permeability coefficient depends on solubility and molecular weight. so it depends on how many molecules are dissolved in the membrane, and the mobility of each molecule within the fluid. most of what we'll talk about is movement of drugs between compartments, across nonaqueous barriers (cell membrane barriers), so we're concentrating on drug movement across these barriers, and that varies in different places. barrier between ECF and ICF is a single cell membrane. if we want to cross from outside body to inside body, from lumen of GI tract to inside body, we must cross an epithelial barrier - also transdermal drug delivery requires crossing epithelial barrier - crossing cells - multiple layers of cell membranes. the epithelial barriers have tight junctions/zonula occludens. so drug transfer must go through the cell, not between the cells. if you want to go from blood to tissue, you must cross endothelial barriers - more complex. several types of endothelial barriers- -continuous endothelium, seen in skin and muscles, mostly tight junctions with some spaces on each side, which are called maculae, and allow passage of small molecules (more permeable than skin) via intercellular permeation. -fenestrated endothelium of secretory and excretory organs ie kidney, with larger holes, more permeable to water and small molecules, but not to proteins. -discontinuous endothelium - large spaces allowing passage of proteins and macromolecules ie in liver, bone marrow -tight junctions similar to the epithelial barrier, with zonula occludens, requiring passage through the cell ie in testis and blood/brain barrier. drugs cross cell membranes by: 1. diffusion through lipids - important. 2. carrier molecules - important. 3. diffusion through aqueous pores in the membranes - not so important. most drugs too big for this, can't fit through pores. 4. pinocytosis/endocytosis - important for macromolecles/proteins eg insulin diffusion through lipids depends on: 1. solubility - expressed as an oil:water partition coefficient. you take a drug, put it in a chamber with some water and some oil, mix it up, and see how much drug is in the oil and how much is in the water -this tells how fat soluble it is. if it is fat soluble it will dissolve in membranes. solubility tells how many molecules are present in the membrane that can diffuse across, or the concentration in the membrane. if a drug has high lipid solubility, it will develop a high concentration in the membrane and then it can diffuse into the cell along a concentration gradient. nonpolar substances diffuse readily - not ions. this lipid solubility is one of the most important factors determinin the ability of drugs to cross membranes - eg, the kinetics of the drug. how well drugs cross GI tract, get into brain, stay in body, etc. all based on lipid solubility. so, the greater the solubility, the greater the permeability. handout p 3 has some diagrams to go along with this. 2. diffusivity - the diffusion coefficient which determines mobility in the membrane. not permeability, but mobility. most drugs have similar diffusion coefficients so this is a minor factor. 3. pH - this is important, because it is going to determine if drugs are ionized or not, since many drugs are weak acids or bases. if you increase acidity, you will drive both equations to the left as discussed before - so the amount of a drug ionized will depend on the pH of the solution it is in, and whether it is an acid or a base (the pKa of the drug). in an acid solution eg stomach, rumen - acidic drugs will be nonionized and will diffuse through barrier readily. basic drugs will be ionized and will not diffuse through the barrier readily because they will not be very lipid soluble (see above). Remember basic drugs are trapped in the rumen because rumen is acidic... ionization also affects rate at which drugs permeate membranes and steady state distribution of drug molecules between compartments. intestine vs stomach - in intestine pH is high, H+ is low, so drug is nonprotonated, charged, low lipid solubility, low permeability. in stomach, low pH, high H+, protonated drug, uncharged, high lipid solubility, high permeability - can cross membrane readily. chart on p 4 of handout - dr robinson will cover something similar to it so davies is pretty much skipping it. the main thing about it is that within each compartment (urine, gastric juice, plasma), the ratio of ionized to nonionized drug is set by henderson-hasselbach - pK of drug and pH of solution. when you go through this exercise you assume that only the nonionized drug crosses the membrane. it reaches an equal concentration in each compartment. so b/w urine and plasma, nonionized drug will be at the same concentration. but, because the ratio of ionized to nonionized varies, the total concentrations of ionized + nonionized will be different. acid drugs concentrate in compartments with a high pH, because it will dissociate and be trapped. basic drugs are concentrated in compartments with low pH. you dno't have to learn this now -you can learn it on friday instead if you want. 4. surface area - the last thing diffusion through lipids depends on. the small intestine is the most important site of drug absorption b/c it has all those villi and microvilli and therefore a huge surface area. even if pH isn't favorable, most drug absorption occurs in small intestine. therefore, anything that increases speed of passage from stomach to SI will increase the rate of absorption of drugs. Carrier mediated transport: special transport mechanisms that regulate entry and exit of many compounds to/from cells. amino acids, sugars, NTs, metal ions, etc. all involve carrier molecules - proteins in the membranes that bind molecules and move them across a membrane. very important for some classes of compounds. 1. may operate passively - passive, carrier mediated transport = facilitated diffusion, large molecules can move faster due to carrier. movement is always in the same direction of the electrochemical gradient 2. may be coupled to energy source - active transport - then, can go against electrochemical gradient. these carriers may be directly coupled to ATP or indirectly coupled so transport is along with something else like sodium ion. 3. involves a binding step to a carrier, same way drug binds to receptor. so the carrier can be saturated. so if high concentrations of drug saturate carrier, amount transported will no longer be proportional to concentration - it will level off. 4. competitive inhibition can occur. second ligand can bind to the carrier, interfering with transport. you can interfere with transport of penicillin in the kidney by using another drug that competes with the penicillin carrier, to keep the drug in the body longer. they used to do this because there was little penicillin around and it was so expensive. it was that or recover the penicillin from patient urine...yikes. Drug Distribution: how drugs get distributed to different compartments and why they stay there. things that affect drug distribution: 1. cardiac output - the higher it is, the faster distribution occurs. 2. regional blood flow -the better an organ is perfused, the higher the distribution of drug to that organ, and the faster equilibrium is acheived. tissues are usually divided into three groups: a) vessel rich - brain, heart, liver - high perfusion, rapid rate of onset of drug action in the viscera. b) muscle - not vessel rich or vessel poor - vessel average. drug uptake is intermediate c) vessel poor - fat, bone, hair, ligaments, teeth. groups with poor blood flow fill slowly. they pull drug out of blood slowly. as they pull drug out of blood, concentration in blood can fall below concentration in viscera, then blood will pull drug out of vessel rich group and put it into vessel poor group. this can pull an animal out of the surgical plane of anesthesia. 3. lipid solubility - fat soluble drugs will be distributed to fat and will be in high concentrations in the fat, making them virtually inert for the rest of the body, having no effect. no pharmalogical action occurs, but a reservoir of drug is formed. this delays biotransformation and excretion. if it is in the fat it can't be eliminated. most drugs do not have high lipid solubility - only some general anesthetics do. therefore, this isn't that important. fat has poor blood supply so drugs get into it slowly. so if you give a single dose, not much ends up in the fat since fat is poorly perfused. so for acute exposure to a drug, fat solubility is only important for a select few drugs. for chronic exposure to drugs, more important - drugs can build up in fat, environmental contaminants can build up in fat, too. then during the winter if animal is starving, fat is mobilized, toxin is released. 4. binding to intracellular elemnts - DNA, muscle protein, bone, etc. digoxin binds muscle proteins. drugs can bind calcium in bones and teeth - tetracycline does this and can discolor teeth. another drug reservoir mechanism that makes drug inaccessible to organs of elimination. 5. binding to plasma proteins - many drugs bind plasma proteins. only the unbound, free drug is active, and this may be only 1% of the total drug. if a drug is bound to a plasma protein, it can't bind its receptor. only free drug is free to diffuse to receptors or into liver or through glomerular filtration mechanism or whatever. see diagrams p 7 of handout. only unbound drug goes to sites of action and elimination. albumin is the most important plasma protein because it is most abundant. binds many acidic and some basic drugs. there are also globulins that bind basic drugs, and alpha1 acid glycoprotein (an acute phase protein present in high concentrations in sick animals) also binds some drugs - so dose may need adjustment in sick animals. amount bound depends on free drug concentration, affinity for binding sites, protein concentration, and competition for binding sites (other ligands competing for same site - drugs or endogenous substances). for most drugs, the binding sites are far from being saturated. Therefore, the concentration of bound drug varies nearly in direct proportion to the free drug concentration. There's a table in the handout which gives %bound for many drugs. Some drugs will approach saturation, so as you increase the amount of free drug, you don't really increase the %bound - there's a disproportionate increase in free drug. he showed us a graph of bute concentrations before and after saturation of protein binding sites. Extensive protein binding acts as a reservoir, slows drug elimination, prevents glomerular filtration although it can still be actively secreted by renal tubule. drug is not accessible for diffusion into hepatocytes, but can be actively transported into hepatocytes. 6. Enterohepatic circulation: many drugs are actively secreted into the bile. once in teh bile they go in the gut. if drug is free drug, it can be reabsorbed into systemic circulation -this prolongs drug activity. digoxin acts this way. if drug is conjugated, complexed to glucoronic acid or something, bacteria can unconjugate it, so it can be reabsorbed into circulation. this is enterohepatic circulation, a mechanism of extending drug action. ---end----