 Okay students, good morning, so this week we are discussing NMR in drug discovery. In the last lecture, essentially I discussed how we can use to develop and drug starting from how we can use the NMR in S3R, structure activity relationship, then looking at the binding site on the protein, we can start developing the drug, so we can find another binding site if possible. Then attach these two ligands to have a better binder, I give you few of the example how we can start from say millimolar binding, KD like a binding strength KD and all the way by joining this fragment we can go up to nanomolar. So this is called fragment based drug design. Then we also looked at how we can look at the ligand site, looking at the interligand NOE and then again we can join these ligands to make a better binder. So this is kind of a development. First is like we have identify which comes in drug discovery, identify a binder by either detecting a ligand or detecting a protein. So once we identify the binder, we want to make it better binder, combining the medicinal chemistry approach and the structural information that we are getting from NMR spectroscopy. So can we combine these two to develop a better drug, a stronger binder, a more potent drug and that is what we were discussing how NMR is helping us in doing this. So interligand NOE actually you can measure the NOE between two ligands that are binding at two different site and probably looking at what are the proximal atoms that are interacting through this NOE and then one can join them to have a better binder. So that is what we looked at and then I give you few of the example how we can use these concepts of SAR, then ligand based, fragment based drug design to find a better binder. So we ended it there, now we will move ahead and we will say that once we design the drug and discover the drug, we have to look at what this drug is doing, is it going through the body where it is going, can we detect like is it going to the right side where NMR can play a role, we are going to now slowly delve into these concepts of I would say drug metabolism and detecting the drug and its metabolites in the body using either in vitro approach or in vivo approach and let us see can we detect it, can we and then this will help us to knowing the efficacy, potency of a drug, how drug travels through the body. So many other aspects can be explored if we combine the concept of drug metabolism and detecting by NMR in per se. So let us move ahead with the concepts. So you know from your like a basic course in the drug discovery and design that the basic property of drug should be something called ADME, drug should be absorbed in the body like any drug that you are taking, whether you are taking a oral drug or injectable drug or subcutaneous drug or intravenous drug, may any of these drugs what are the mode of injection it is, it has to be absorbed in the body, the first and most important thing is absorption. Once it is absorbed it has to distribute throughout the body right. So distribution should happen as systemic distribution it should reach to the location where it has to act like many of the drug will take through oral injection. So when we take a tablet or capsule we just take it through mouth, it has to absorb in our system, then it should be distributed and it should reach to the point of action right. So that is a distribution. After that once the action is done this drug is not from our body right, it is a foreign particle you know biotic or so we it has to be metabolized, it should be degraded into smaller pieces and then finally it should be removed from the body. This is called ADME and this can this is actually required to understand the toxicology and this field is called like a toxicology that is studied in human or animals. So it should not produce any toxic effect that is the primary requirement we are taking a medicine for being cured, it should not produce toxic thing. So you have to identify whatever metabolites are produced by the drug metabolism should not have any like a or minimal toxic effect. We cannot ensure for no toxic effect but it should be very very minimal toxic effect otherwise drug has to be recalled and that drug cannot pass through right. So these four points has to be very well taken care, it has to be absorbed distributed metabolized and excreted that is called ADME. So here in the cartoon responsible, so first thing is like you can even take it liberation like it is the active pharmaceutical ingredient should be liberated from the formulated capsule or a tablet or wherever it is and then it comes to our body by absorption, it gets absorbed, it distributes through our circulation system and that is a distribution, it reaches to brain, it reaches to head, it reaches to hand all those right and then metabolism so it should be metabolized and finally it should be excreted through urine or through ficus or somewhere or sweat but it has to go out of the body that is the typical property of any drug that is there. So this whole concept is called pharmacokinetics. The kinetics of a pharmaceutical molecule, pharmacokinetics that is what this study is like if you design a drug, you are administering drug how it is going through our body, so you are taking a medicine suppose through oral route, it should go to your liver, to intestine and all those and then like that will be and how it should be absorbed, so it should be absorbed through your tissue and cells, absorption how it will get absorbed then how it is broken down, so most of the breaking down happens in liver we will look at the little more detail how it happens and finally how it is distributed, where it goes, what are the transporters for these drug molecules? So here you can see there are various transporters and all one of the obvious transporters is a blood, so blood can take because blood goes everywhere, so blood can take the drugs throughout the body and finally after metabolism it should get excreted, so that is total concept of these drug journey from the administration to the excretion is called pharmacokinetic. So drug metabolism and how it can vary among the patients in a drug response that determines a lot of things, so when you go to a doctor a physician, he prescribe drugs based on the characteristic of a medicine and the probability that has been obtained from reliable and reproducible clinical effect what result it will produce, so that is the basic concept that physician has, there are some already reliable and reproducible results obtained in a clinical trial that this drug should act in this particular manner with some variation and that knowledge doctor has physician has, when one new person goes to physician he understand the characteristic of a drug and the probability that has obtained from clinical trial, so he prescribed a drug but as you know all of us are not similar. Our system response is different, therefore it is expected that there will be difference in the drug response among the patients that goes to a physician and that is obvious that is common because we are different. Genetically we may be same but our protein expression level transcriptomics like transcript level we are different, our protein may be same but their action like how fast or how slow or how pronounced they work are different, therefore the drug response in different patient can be different and that often leads to challenge in optimizing a dose, how much a dose should be given to particular individual depending upon the severity of the disease one dose will suit to a person a particular person the another dose may suit to another person. Now, commonly nowadays what is happening that generally doctors prescribed one dose for adults, one dose for child you know if you know from your common language child has a lower dose adults had one dose but let us talk even adults, adults like all adults are not same, so I like if it is intuitive that if one has to prescribe the medicine or a dose should be person specific rather than a like a blanket prescription. Now, how to get that person specific response for that that is called now the field will be coming is called personalized medicine. So, every person should be given in a particular dose form and that can come only if we have detailed a study how a person is behaving for a particular kind of metabolism drug metabolism. So, that will dictate about the dosage the particular kind of medications should be given to a person. Now, for doing that for knowing that actually lot more studies required a larger sample size has to be taken person specific dose should be determined looking at the person metabolism and therefore drug metabolism becomes of paramount importance for designing and optimizing the drug response in a particular person. Then another major hurdle that major drugs are effective in only say 25 to 60 percent of the patients it is not. Now, this is the question one can ask why drugs should by all of us are human. So, should be effective on all of us but that does not happen one drug works for a person fantastically it does not work for another person. Now, that is that is the paradox that still exists and therefore it has to be studied in detail. And here comes the NMR which can study person specific the drug metabolism by taking some of the body fluid like in vitro we can use the body fluid like a tissue extract or a urine or a blood, serum or plasma or one can even take the intact cells or biopsy or perfused organ or NMR can even offer to study the metabolism in whole like a animal model or human subjects. Combining probably MRS and MRI, MRS is magnetic resonance spectroscopy and MRI is magnetic resonance imaging. If you can combine even you can look at the feet of a drug inside the body. So, there the field is going towards developing, but in vitro can be done quite easily taking urine, taking serum, plasma or tissue extract, biopsy sample and all those. And it offers the unique probability, NMR offers the unique ability to do these kind of a study in vitro and in vitro simultaneously. So, without perturbation or taking just body fluid without changing much in the body fluid you can detect what is the fate of a drug that is administered. And that will create a wealth of information to leads towards the personalized medicine that is the future subject in the medicine field. How you can tune particular, tune a drug or a dose for a particular patient that is going to come in future. So, what happens for the drug? So, when drug enters the system, when drug is entering the system actually one thing I said that the its availability is only like a very low. So, why it is low? What happens? When it go to like liver the proteins are there which starts cleaving. And there is a heme protein called cytochrome P450 that plays a key role in the metabolism of drug or any genobiotic. This is a heme protein, this is the structure of a protein, there is a channel here, lots of like here is N-terminus, the prosthetic group where actually heme binds and here is the C-terminus. So, this protein cytochrome P450 it is a important protein, it also involves in biosynthesis and degradation of indigenous component such as steroid, lipids or vitamins. It is also responsible for metabolizing any genobiotics that drug that we are taking. Therefore, what can happen? The P450, cytochrome P450 action can be differential in a different person. Therefore, the drug that we ask the question of why one drug is effective in one person it is not effective in another person can be understood that the protein expression can be different in different person. And therefore, the available drug that is there can be also different because of action of this protein called cytochrome P450. So, cytochrome P450 plays an important role in chemical toxicology. So, again this is the structure it is a heme binding protein you can see. Therefore, the heme binding site and in a oxidation reduction reactions coupled with NADPH cytochrome P450 actually does couple of oxidation reduction and that helps in metabolizing any genobiotics. Interestingly in human there are 54 cytochrome P450 genes and they are responsible for different kind of metabolism today we will be highlighting some of those. So, in liver which is a major site of the metabolism this cytochrome P450 exist. So, this protein cytochrome P450 start metabolizing drug and as we know that once you take this drug here once you take this medicine it goes and goes through liver. Here is our metabolism happening and then it goes for absorption also and at this place our cytochrome P450 is sitting. So, cytochrome P450 mediated metabolism happens, but in erythro site RBC there are epithelial cell or even in the epithelial cell of sorry small intestine that are potentially important site for metabolism can happen. So, major site is our liver it can happen in intestine also these are the important site for drug metabolism like one of the cytochrome P450 is CYP3A which is found in erythro site RBC structure is here. Now this also can do metabolism. So, you can see first is liver it can be in RBC it can be in intestine epithelial cells of intestine these are the potential site where drug can metabolize. So, about 40 percent of commonly used drug is only available because that gets metabolized by limited absorption or first pass metabolism. First pass metabolism means like it has to pass through metabolism and then it can be absorbed. So, now the drug whatever we are taking is not 100 percent available it gets metabolized how much depends upon a person where it gets metabolized in liver in RBC in small intestine. So, here is the first pass metabolism that happens after oral administration of a drug. So, for this I have taken from new general of like New England general of medicine you can see if you are taking the medicine through oral administration it is entering to the liver. So, this drug is called phylo-depine which is actually used for treating the high blood pressure. So, essentially this drug is a calcium channel blocker and what it does it works by relaxing the blood vessel. So, heart does not have to pump very hard and that is how it is out the blood pressure right. So, this drug is taken suppose this drug we are taking 100 percent phylo-depine dose now this is going through the liver ok. Now, here is liver sitting then goes to a small intestine and all those and it gets metabolized. So, here this drug is going 100 percent after metabolism here in RBC we have this protein called CY34A it starts getting metabolized. And then if you look at the drug percentage that starts getting reduced from 100 percent here is a 45 percent here 15 percent, 13 percent or so somewhere it is 19 percent. So, in gut lumen in erythrocyte this is 90 percent or so. Finally, when it comes out and goes for a circulation it is 15 percent or 45 percent. So, rest about 85 to 60 sorry 55 percent is lost in the metabolism in liver small intestine and RBC. Now, that is called bioavailability how much drug is available for circulation and that has to reach to the point of action. So, 100 percent it starts and you can see that about 45, 55 percent to 85 percent it gets lost, it gets metabolized. So, this guy if you take it phylo-depine you are taking some 100 percent, but it is available only 15 to 20 percent. Now, that requires investigation in a person specific. So, you cannot because this action of these proteins can be differential in different person. So, you cannot recommend or prescribe one dose for all it will not fit. So, for one guy it will be very effective one person it will be very effective for another person it will not be so effective. So, probably you need to readjust the dose so that that person also gets the significant amount of bioavailable drug to reduce the blood pressure and therefore, you see the doctor keep changing the dose right. So, looking at the response from you it keep changing the dose it can start from lower dose then depending upon how you behave they can increase or decrease the dose if required they increase if required they can decrease the dose and that is the iterative process goes on and that is determined by the bioavailability of a drug. So, here is the detailed description of the same we are starting from 100 percent and then in some of the tissue rathrocyte and the intestinal tissue it gets metabolized and finally, we have only 45 percent right. So, some of the commonly used drug which has a low bioavailable you know many of these drugs and what are the metabolizing enzymes that do and what is the percentage bioavailable I am showing you. So, one of the drugs say let us say aspirin right we many of us take for headache aspirin enzyme is called esterase and you know it is a easily soluble drug right. So, if you put it in the water it is easily soluble you take it through oral or you dissolve in a water just drink the dissolved water it is 68 percent one of the higher one. Now, another one you can choose any of these drugs phyllo dopine that we just now we looked at. So, only 15 percent it is a like let us say heart yeah heart disease does drug. There are many other drugs dichlofenic these are these enzymes that are involved and 54 percent. So, if you look at many of these drugs the bioavailable active pharmaceutical ingredient is about 50 percent or less than that. So, one important area of research that is coming up how you can increase the bioavability of a drug for that also one has to do a systematic investigation looking at what is the fate of a drug what is the fate of metabolism that happens and how much drug is available for action. So, the active area of research to increase the bioavailability how we can increase the bioavailability right. So, this is an important area of research. Now, these guys the paper that I am citing it actually they come up with a interesting thing. So, like a consequence of inhibition of first past metabolism if you do inhibit the first past metabolism where our cytochrome C cytochrome sorry P450 is sitting if you inhibit that you can increase the bioavailability. So, for the same drug that we were discussing the phyllo dopine if it is taken just in water you look at the bioavailability this is the time here in hours and this is the percentage or the plasma concentration that is reaching to the blood plasma concentration in nanomole per liter. So, if you look at it increases 0 hour when you take the drug with time it increases goes to about 15 percent and then slowly slowly decreases in 8 hours it becomes about 1 percent or even less than that. But if you take this drug with a grapefruit juice grapefruit juice that inhibits the first past metabolism and you can increase the availability of this drug up to like about 30 percent and even after 8 hours this remains about whatever we had earlier right 8 to 10 percent. Now, just by taking this drug with a grapefruit juice there can be another substitute but this paper describes about grapefruit juice. Now, how that response happens because finally you are giving and increasing the bioavailability does it changing the heart rate. So, if you look at here heart rate plotted bit per minute right. So, how heart is you are pumping because there was a pressure on the heart that is why your one is taking the blood pressure drug. So, if you look at the grapefruit juice and water in 8 hours the grapefruit juice is slightly behaving better than the water and even after 8 hours both are doing fine. So, I think you have improved the bioavailability and that is how heart rate is slightly performing better in the grape grape when you are taking with a grapefruit juice. Another is blood pressure does it maintain the blood pressure. So, actually if you look at the grapefruit juice actually it reduces the blood pressure it brings in the range of 115 rather than going to more than 120. So, grapefruit juice not only increases the bioavailability but also it decreases the heart rate and blood pressure in mm also like a diastolic also becomes better like a the higher one lower one. So, both the blood pressure basically systolic and diastolic actually improves when it is taken with the grapefruit juice. So, blood pressure and heart rate were measured while patient is where standing it is not asleep. So, in a active condition one measured and what they found that when this drug is taken with a grapefruit juice essentially it increased the bioavailability of a drug because it inhibited the first past metabolism and that is how the effective concentration is there. Now, the question is how this was measured right. So, how this was measured. So, one of the analytical techniques could be used for measuring it and that is what we are going to to discuss how NMR can play a role in measuring the effective concentration of a drug. Now, so what actually cytochrome 3A4 mediated metabolism and did for drugs subset. So, when you take a drug any of this drug you are taking it, it goes and binds to some of the protein complexes co-activator comes and there is a core is the pressure this is our cy34A and that basically plays an role in the metabolism. Now, one once you take a kind of a inhibitor that probably it inhibits. So, that can increase the sorry decrease the activity of the one of these drugs and essentially if you inhibit or reduces the activity of this protein then the bioavailability can be increased. So, many of such things such things can be tuned for increasing the bioavailability or you can formulate your drug with something else. Now, active research is going on how to formulate co-formulate your drug with something else which increases the bioavailability. So, I will stop it here. So, the on the metabolism part, but if you look at the plasma phthalodoprene concentration in nanomore per liter you can see the patient in a healthy subject you can see the concentration of plasma phthalodoprene was quite higher and the patient which has increased activity of this particular enzyme of cytochrome P3A4 the concentration of this drug was very less right it is one here one nanomole and here we have a 6. So, that means the person who has a more active protein of Cyp34A actually their drug is not that effective and in healthy subject it is quite a bit. So, what we need to do now if you want to really treat the heart disease the blood pressure or something you have to also temper with this enzyme activity. So, that whatever drug is given it is not metabolized and the effective concentration should reach to the point of so that heart does not have to overact right. So, that is what it is done by grapefruit juice and these all information I had taken from this prestigious general of New England general of medicine you can refer to this general. So, this is called co-formulation and increasing the bioavailability. In the next class what I am going to discuss with you that how if this is the problem how NMR can play a crucial role in understanding the drug metabolism at at various stage using in vitro and in vivo approach. So, let us first start with an in vitro approach and then we can go to in vivo approach. So, I will stop it here and looking forward to have your questions in the next class. Thank you very much.