[MUSIC] The subject of this module is a man who has a second heart attack a month after having a first heart attack. And the major points are that we're going to talk a little bit about bioactivation of a prodrug. Mechanisms in general underlying variable drug effects, the question of whether genetics or functional tests are better to assess drug action, and then we'll start to talk a little bit about the question of the level of evidence that we need in order to introduce genetic testing into the ordinary flow of health care. So the story is a 62 year old man who's admitted with acute coronary syndrome. He undergoes cardiac catheterization, is found to have a relatively tight mid right coronary artery lesion with thrombus. It's successfully stented, he's discharged on clopidogrel, 75 milligrams a day. He's admitted three weeks later with recurrent chest pain, and at catheterization, he has in-stent thrombosis. So what are the potential explanations? So first of all, why do we use clopidogrel in a situation like this? Well, we have a randomized clinical trial, we have a number of them, this is one of the larger ones that compared placebo to clopidogrel in patients who presented with ST segment elevation myocardial infarction. And over the course of 30 days, the incidence of a composite endpoint of recurrent myocardial infarction, stroke, death or instant thrombosis was higher, as you can see in this graph, in patients randomized to placebo than those randomized to clopidogrel 75 mg a day. So one take-home message is that everybody who gets a stent gets clopidogrel. The other take-home message is that we can't guarantee that there will be absence of events on clopidogrel. So that's the fundamental biology. The most important question that we have to ask when we first see the patient is did you remember to take your medicine. Non-compliance is a huge problem, it may overshadow any genetic effect or any other kind of variable drug effect the patient isn't taking the medicine, the patient isn't going to have an effect. And there's many reasons why a patient might not take their medicine. They might forget, they might be prescribed 20 different medications a day and get confused, they might not be able to afford their medicines, they might not think the medicines are all that important. We have case after case of patients prescribed blood pressure lowering medicine and after a month they stop because after a month their blood pressure must be cured. So people need to understand why it is they're taking the medicine, how important the medicine is and among the many medicines they take which ones are the most important because sometimes people have to make choices. But compliance is a huge issue around the practice of medicine, and, of course, the practice of personalized medicine. So clopidogrel, let's talk a little bit about the way in which clopidogrel is gotten rid of in the body. Clopidogrel is an example of a prodrug, a drug that compound is not itself active but requires metabolism to be bioactivated. Most clopidogrel actually undergoes metabolism by esterases and never reaches the systemic circulation in an active form. The bioactivation occurs in the liver and for a long time, the exact bioactivation pathway was not known and a number of liver enzymes were implicated. It turns out the most important enzyme for clopidogrel bioactivation is CYP2C19, one of the members of the cytochrome P450 superfamily. After clopidogrel is bioactivated, it then interacts with ADP receptors on platelets to inhibit the action of ATP to cause platelet aggregation. So that's how the drug works, and that's a basics of its clinical from oncology. There are important and common loss of function variance in CYP2C19 and they're shown on this slide. The most important and common one is *2 and depending on what ancestry you look at, *2 is quite common as you can see on the table. *3 is another variant that is quite common in Asian populations but rare otherwise across the globe and then *17 is an interesting variant. It tends to increase the activity of the enzyme so that patients who have the *17 variant you would predict would have greater anti-platelet effect with clopidogrel. And that's something I'll come back to at the end of the talk. And that's common in European and African populations much less common in Asia populations. So we call people who have two star one alleles as wild type or reference genotype. And then there's the intermediate genotype with one star one and one loss of functional allele and poor metabolizes and then the ultra rapid over on the right hand side. As an example we at our hospital have been genotyping patients for CYP2C19 for a number of years, and here are the results in. Over 13,000 patients in a population that is largely Caucasian but about 15% African American. The overall incidents of homozygotes, for star two star three, and then three other alleles that are quite rare but we see them anyway, is 2.7% for the homozygotes or the compound heterozygotes. 19.4% for individuals who carry one of those loss of function alleles and then the remainder approximately 78% or so have no variant that we detect. This is the results of a genome by dissociation study examining the outcome of clopidogrel induced inhibition of ADP-stimulated platelet aggregation. These are 400 patients who underwent ADP stimulated platelet aggregation in the absence of, then in the presence of Clopidogrel. This is an X vivo test and you can see that the results show that there is. Clearly one dominant genomic locus at which there are common variants that appear to drive variability in this trait. Interestingly, the way this study was done was in a large group of Amish subjects. They're all related to each other, and the relatedness allows one to estimate the heritability of the trait. Relatively accurately, so the heritability of this trait is large, 73% of the variability in the trait is heritable. That said, the signal that you see on the GWAS is right in the CYP2C19 locus. Locust and in fact when the authors corrected foe the fact that there were CYP2C19*2 they included that as a covariant that signal now goes away completely, what that means is that the entire signal that you're looking at is a sub CYP2C19*2 signal. Interestingly, only 12% of thew variability in the signal is accounted for by sub CYP2C19*2. So if you're a pessimist, you say, well, there's a huge amount of variability in this trait and a little bit of it is due to CYP2C19*2, and a lot of it is due to something else that we don't understand. If you're an optimist, and geneticists are optimists, they say well, It's extremely unusual to have a single snip that accounts for 12% of the variability in any trait, be it cancer susceptibility or acute myocardial infarction or whatever. So the genesis would say this is an enormous signal, and if you infactor in known clinical factors like BMI and lipids. You can increase the explanation for the variability, but you still have a large chunk of the variability in this trait unexplained, and that's a pretty common scenario across the genome-wide association world. So people have gone on to look relatively retrospectively at the outcome of clopidogrel therapy as a function of genotype in very, very large clinical trials. These are meta analysis showing the results of the individual trials, they're confidence intervals and an over all. Estimate of the affect size among subjects carrying one copy of a loss of function allele. And that generally means CYP2C19*2 at least in the non Asian populations and the point estimate is about with an odds ratio about 1.5 for a recurrent event. That means that there's 50% increase risk that patient taking clopidogrel will have an adverse event at the normal dose after receiving clopidogrel, like our patient did. Patients with two copies of varied alleles, there are fewer of them, so the point estimates have wider confidence intervals. But the odds ratio is 2.8, so that's pretty high. So the question is, do you in fact test everybody beforehand? The optimist would say well, I want to know whether my patient is a star 2 star 2 perhaps even a star 1 star 2 because I might choose a different drug or I might choose to increase the dose of the drug. The pessimist would say well that's a pretty small effect. Keep on telling about odds ratios of 100 and 200 and those are actionable. Here's an effect size that is 50% so why should I get excited about that? There's so much variability you haven't explained maybe what I should be doing is testing the way in which platelets aggregate during therapy. It turns out their patients who get clopidogrel and you test their platelets, and their platelets still aggregate just fine as though the drug weren't there and there are patients who aggregate just fine. So maybe instead of measuring genotype I should measure net drug affect downstream. And there are such tests that's controversial exactly where they fit in and how good they are predicting outcomes. The other thought is, well let me do the genotyping among patients who are star one, star two, I'll just increase the dose. And there's a little bit of support for that approach. Not perfect, but some support for that approach. What's interesting is, if you try to increase the dose in the stars two, star twos who have no CYP2C19 activity, or very little activity. You can't increase the dose enough to overcome the genetic defect. So one strategy is to say, well, the star two, star twos, certainly, have a defect and maybe they should be considered for an alternate therapy. Star one, star twos, maybe, maybe not, and that's the way many people approach that now. So clopidogrel is a Pro-drug and it is bioactivated, and this bioactivation occurs through a single pathway. So the problem with a single path way of course is that if anything interferes with its activity. Then the Pro-drug accumulates and the active metabolites don't get made and the pharmacological effect is missing. So a Poor metabolizer trait is an example of how you might lose that bioactivation. Are there other ways? Well, it turns out there are drugs that inhibit CYP2C19. The most commonly used inhibitors are the proton pump inhibitors, notably on Leprosol. And these are data, retrospective data, from a large pharmaceutical benefits provider that looked at the incidents of recurrent events after an initial prescription of clopidogrel for an acute coronary syndrome. And what you can see is that among patients who ended up on clopideogrel at a PPI there's a higher incidence of events compared to patients on clopidogrel. This is retrospective, it's not randomized, but it still suggests that there's a consistent signal. One question you might ask your self is well, maybe that's just because PPIs make people worse. PPIs have some affect on platelet aggregation, but it turns out they had a group of people that were only on PPIs. They didn't seem to get worse than people who received neither drug. So the answer is, it looks like from this retrospective examination, that there really is an interaction. Another group of investigators then did a very prospective study, involving about 3.5 thousand subjects followed for a year, after being randomized to clopidogrel + PPI vs clopidogrel + placebo. And if you look very, very carefully, there are two curves there but they really are super imposed So these data which are prospective and randomized argue that there is very very little, there is no affect of PPIs. The criticism against this study is that the event rate is quite low compared to what we saw In this study here for example. And there's a reason that this study wasn't continued longer it had to do with funding. But at this point it's hard to argue very strongly that the PPIs are absolutely counterindicated. But the weight of the pharmacologic evidence would suggest that at least very PPIs that are very potent CYP2C19 inhibitors probably aren't the best thing to use for patients receiving clopidogrel. And maybe other PPIs that are weaker CYP2C19 inhibitors are better to use, the strongest inhibitor is omeprazole. So we have the situation which a pro drug is susceptible to not working. Because it has this property that it requires activation, there's one pathway, and anything that interferes with that pathway will then result in failure of drug effect. We also have the situational course with the CYP2C19*17s that those patients may have excess effect. This is relatively small and quite controversial, with clopidogrel there are some who think the incidence of bleeding is higher in patients who carry this allele, there are others who say the signal is very, very small, those are complicated patients. And most people are not paying much attention to the ultra-rapid metabolizer trait, certainly not reducing the dose because of that. So what are the explanations that someone who comes in with an apparent lack of drug effect presents to us? In this particular case, the explanations were this is biology, the drug we have isn't perfect at preventing the effect that we're looking for. The patient might be non-compliant. The patient might have varied genetics that makes them not likely to respond to clopidogrel. The patient might be taking an interacting drug. And these are generic explanations that apply to many, many situations In which we find ourselves in modern therapeutics. And so the bottom line is that there is multiple mechanisms that can underlie variable drug effects. Pro-drugs are a special problem because they have this high risk property that they require activation through a single pathway and if that pathway is variable at all that there's a problem. And then there's the generic question that this story raises and that we're going to come back to over and over again and subsequent modules. And that is, exactly how much evidence do you really need before you start to say, I need the genotype or I would like to have the genotype or wouldn't it be interesting to have the genotype in this patient in order to guide my selection of drugs. There are other ADP platelet receptor antagonists on the market. They're a little more expensive right now, because clopidogrel is generic. And they have the advantage that they don't have genetic stories, they had the disadvantage of cost, and they had the disadvantage of other side effects as well. So a physician faced with this kind of patient may choose to use an alternate and the genetics may drive them in that direction. We'll talk more about those kinds of things in subsequent modules. [MUSIC] >> [APPLAUSE]
