 Good morning students. So, this week we are discussing how we can use NMR in drug discovery and then like in last lecture I was discussing how we can use in understanding the drug NMR in drug metabolism right. So, we discuss that drug metabolites and it passes through various body fluids. We can take those body fluids and in vitro detect the level of drug and drug metabolites. Some of the obvious body fluids are urine, the plasma serum like a straight it can be speak many of these body fluids can be used for understanding the drug metabolism. The two famous one are plasma and serum and also urine that can be drawn. So, NMR can you give NMR can give you quantitative assessment of drug metabolites. Also we looked at what are the nuclei that can be used for detecting drug metabolism. The famous one can be proton and it can be carbon then the best one appears to be fluorine in terms of sensitivity, in terms of the dispersion and the relaxation time. The problem with proton was like it is overcrowded by already the intrinsic involvement that comes in body fluids like urine. So, getting the clear signal from drug which is very low concentration is can be often difficult. So, you need to combine with a 2D experiment when you can transfer the polarization from proton to X nuclei and detect on X nuclei to have a more clear understanding of drug metabolites. But those were all in vitro experiment. Can we also think of detecting the drug metabolites in in vivo system right. So, in what is the good part of in vivo system that like one can measure the drug plasma level drug level in plasma, but that does not always reflect the drug concentration at the receptor site right. So, plasma is circulating right this is in circulation how much drug is circulating that is what we are measuring when we measure the plasma and drug concentration. But what happens the effective concentration that is reaching to receptor site that will not be directly reported once we measure the drug from the plasma right. So, we need to have a method that can allow us to measure the concentration of a drug and their metabolites in C2 and that will be very useful right. We can measure wherever the drug should go and what is the concentration of that drug. If we can measure that that will be wonderful. So, these experiment can be done if we actually do the in vivo study of drug metabolism. So, the good part of the in vivo study of drug metabolism that measurement of drug and metabolites can be done in the target organ wherever drug is going can we directly measure from there right. And the another good part that it can be repeatedly done on the same patients right. So, reproducibility or the variation that will come from batch to batch or measurement to measurement can be also accounted for. And this is non-invasive you are not taking anything out of the body. But just using some probe which is NMR based probe in non-invasive manner to detect the drug concentration at the site of its action. So, that is the good part of in vivo study of drug metabolism. Let us go ahead and look at how we can do that and what we can do it. The some drawback of this in vivo drug metabolism that generally it is very insensitive like it is a sensitivity is 1 to 10 times less than in vitro concentration. The another big problem is like since these drug metabolites are not freely tumbling in the solution. So, that means there is a unrestricted mobility because of the because of surroundings it come can be coming from different proteins, lipids, muscle whatever it is. And since the mobility is restricted. So, you can understand that lines is going to be brought. So, if in vitro we have a sharp line in vivo we can have a broad line. That is the one of the limitation of doing experiment in vivo because of restricted tumbling or restricted mobility. Now and because of the sensitivity is low and also the lines are broader you require a longer data acquisition time right. So, many like a many scans you have to do. Now another problem can be like a the signal can come from various anatomical origins. It will not only come from the drug in some cases like a fluorine which is a unique nuclei that is not found in the body. If your drug is fluorine level then you can get a signal exclusively coming from fluorine. Otherwise if you are detecting say proton or carbon-13 it is obvious that signal will come from different anatomical origin molecules right. So, and the another problem will be resolution will be poor. So, chemicals chemically similar compound will be difficult to distinguish. So, the good part is in vivo detection of F 19 and that can be actually detected and it has been done extensively of 5 fluoro uracil which is a cancer drug and its metabolite has been estimated around 0.05 millimole per gram that is what minimal you can detect it from in vivo experiment right. So, some of these nuclei are really beautiful for detecting the signal of drug or drug metabolize in vivo setting right. So, that is the application we can think of F 19 based and we can look at what else we can do. So, let us start with a F 19 based nuclei. So, just to remind you why F 19 if you go few slides back in the last lecture we had just given you the idea about F 19 I will just repeat few of those. So, F 19 the first good part of this it is a half nuclear right it is a spin angular momentum is half it is relatively narrow line sensitivities very good like about 83 percent of proton it is a 100 percent natural abundance and the most important part that it is a short relaxer on time T 1 that means you can repeat this experiment very fast and the spectral width is very large. So, you can distinguish the signal coming from different origin of F 19. So, because of all these good part that F 19 has it can be also used beautifully and exclusively in vivo setting ok. So, F 19 study were used in anesthetic and psychoactive or antineuplastic drugs and like what these drugs has the F 19 nuclei and it was used for detecting in vivo. So, the distribution of anesthetic in the brain can be used or even the pharmacokinetic of their elimination are used and how it is being done it is still subject of controversy, but it can be used like you can detect it where it is going and how the pharmacokinetic is playing role. So, the feasibility of in vivo F 19 NMR in human has been demonstrated one of the good example of this drug called fluotaxine which is widely used for antidepressant drug. So, this F 19 drugs were used and then you can see that this is the signal coming from. So, just to give you an perspective. So, once you administer this drug in the in vivo setting it can make some adduct or it can it can do some chemistry to make some other compound right it is it will also get metabolized it can mix up with something. So, this drug called fluotaxitine actually make an product called nor fluotaxitine and these two are present in this in vivo setting. So, you can see that there are two peaks coming in the in vivo setting coming around say 61, 62 ppm one is for fluotaxitine and another is for nor fluotaxitine and if you detect the same thing in vivo you get actually the two peaks and these are the two peaks reflected. So, the first it gives confidence that we can get the clear cut two peaks. If you look at this is these are little bit shifted than this. So, the chemical shift can be different because you can see you can imagine that environment will be different and that is how chemical shifts are different. But good part is that since there are only two molecules which has a fluorine we can conclusively confidently detect this drug in in vivo setting. Now, if we have done that then what next we can do? So, we can do the NMR study on this fluorinated drug some like suppose 5-fluoro-uracil. So, yeah, so 5-fluoro-uracil F19 can be used because of various reasons. So, first one can hydro's administration can be done of these compounds and then fluorine remains intact right. So, fluorine is not mixing or it is not changing. So, even during the biotransformation even during the metabolism F19 remains intact and that wherever they go in the metabolic cycle you can detect it take the signal coming from the various body fluid and you can detect it or even in vivo setting you can do that. So, fluorine atom remains intact during the biotransformation and F19 signal can be therefore displayed over a large spectral width without any overlap and one can detect it. So, you can see here we are getting the fluoro-uracil and fluoride ion, but some other product we are also getting like a fluoro-melanoic acid semi-aldehyde or fluoro-acetyldehyde. So, acetyl these are mixing up and making some kind of another secondary metabolites, but still the signal remains for fluorine signal remains intact. So, you can get this idea where the fluorine is going, what kind of reaction it is doing and then what kind of product it is making and one can detect it right. So, actually one can even do the fluorine 19 spectrum for the per fluid state body weight. So, you can take the F19 here again the 5 fluoro-uracil, the fluoride ion and various other products that are coming. So, this experiment was done, what the drug was doze to a rat and then his actually liver was isolated and perfused and that it was found that the various metabolites that are coming from here from the rat liver. The metabolites of a few that fluoro-uracil is not a fluoro-beta alanine, but metabolites continue to lead to new metabolites like a fluoroacetyl or fluoro-melanoic acid semi-alcohol. So, these are metabolites that are coming up. So, what we are learning here we were injecting this 5 fluoro-uracil and then it is going in the body doing the reactions and making new products. So, that new products we are detecting from say rat liver. Now, we are trying to probe the chemistry goes on when the this drugs are administered what new products are made. So, all these products we can essentially we can detect it. So, you can see here is the concentration of FU, here is a fluoride ion and whatever various products that it made one can detect it and understand what new metabolites are made like a fluoroacetate or fluoro-melanoic acid semi-alcohol these all products are made. So, essentially in view of fluorine 19 NMR has been used to monitor the FU metabolism in the liver and it can be also say used for like understanding the metastasis of colorectal cancer patients. So, essentially what you have to do like a patients were treated with a continuous low dose of intravenous infusion of FU until the point of of refractory of the disease and then you are doing something like a putting the patients doing the localized MRS which is called magnetic resonance spectroscopy and detecting the level of FU that is going on and then you probably inject some intervention like interferon alpha and then also you looked at how it is modulating the FU activity. So, in one case you are just putting FU looking at the signal that is coming from the patient then subsequently you are dozing with interferon alpha and looking at the activity of FU how it is changing. So, these experiments were done. So, F19 spectrum of the liver metastasis from colorectal cancer a patient treated with 5 FU were taken you can see the signal that is coming 5 FU and some catabolites that is coming and after treatment the catabolites that came out was alpha fluoro beta alanine. So, you can see here is the catabolite. So, after some days actually the the catabolites that coming out prominently. Now, you are what next step was done interferon alpha was given to that patient and again looked at what kind of of of the response it is coming. So, 5 fluoro uracil plus interferon alpha was given and you can see here was the anabolites that sorry that started and you are getting the 5 fluoro U and plus catabolites. So, you are essentially saying that how the intervention changes the metabolism in a quantitative manner if you are detecting these signals coming from the patients. So, this is the good part of doing the drug metabolism in vivo. So, one can detect what signals comes out and the what is the pathophysiology or all those will be understand by doctor. So, it helps actually medical practitioner to understand the metabolism that goes on a patient and they can tune their drug dosage or drug regime to better suit to a patient. So, that is what it helps actually understanding the path that drug will take the how metabolism is getting affected in a non-invasive manner. So, essentially patient is going in this machine doing the MRS and giving you the response from the localized place of liver where metabolism happens and then doctors can decide what needs to be done in case of the altered metabolism. So, that is a good part. Now, this in vivo detection is called say MRS. So, you can put the patient in MRI scanner and then do the MRS magnetic resonance spectroscopy. You can detect various metabolites that are coming. So, here I am showing you from brain various like a choline, keratinine, GABA, glutamate all these signals can be detected. So, this gives us confidence that anything that changes in brain we can do localized spectroscopy called MRS and detect what kind of altered metabolism we have. So, I will give you some example of altered metabolism which can be detected using MRS. So, what essentially it is done one has to put the whole organism or whole animal in the magnet and you apply some magnetic field NMR radio frequency on this say right and in the magnet you are saying. So, without any magnet all spins are randomly oriented with the magnet spins are now aligned and that gives you the signal. That is what we know from this course that one has to put the observe whatever being observed in the magnet and that gives us signal. So, if you are putting whole right and taking the slice of his brain like a slice means like a voxel or something like that in MRI scanner getting the signal exclusively coming from there one can know what is going on the rat brain or even for an example any organism. So, one can combine this MRI with MRS, MRI gives you image like you know this is not a course for MRI, but just give you very brief idea you can get the magnetic mapping of the brain using MRI magnetic resonance imaging. So, you can get the image and from the section of that image if you do the MRS magnetic resonance spectroscopy similar like experiment that we did that we had discussed in this course. Take the signal do the Fourier transform you get the MRS spectrum like suppose from this section of the brain you can get all the metabolites that are coming like a choline the glutamate and all those and now this actually is essentially used in the drug metabolism, right. So, if this is a normal patient this is the response if some drug has reached here the response will change and that is what we will be detecting it. So, MRI and MRS combined gives the whole magnetic mapping of the body and what kind of metabolism happens. Similar thing can be done say P31 MRS of the human liver if you take the human liver take a phantom and transverse MRI around the liver you can get various phosphorous containing compounds like a beta ATP, alpha ATP, gamma ATP, NADPH all these signals will be there phosphocholine, phospho star, PI many of these can be detected. Now, suppose I am giving one example. So, suppose one person is exercising right very vigorously and you take the MRS of that person. So, some of these energy currency will be metabolized. So, you will see the response or the ATP concentration will be going down. Suppose, you want to give a drug and look at the metabolism of that what will happen? So, suppose this drug is phosphorous or it enhances the energy metabolism, you will see that the ATP currency the energy currency like ATP-ADP their concentration will be going down. So, that means, if you combine this in vivo detection one can find it out how the concentration of each of these metabolites are changing in case of exercise in case of drug administration and all those. So, combining the in vivo detection of these signals helps us in understanding the metabolism as well. Another example I want to show you what happens in a rat brain. So, you can see which the rat brain which has a seizures you can see lots of this energy currency showing very high signal compared to the baseline. One can have that these concentration or these signals looks slightly higher in case of the seizures brain. So, that means, brain becomes hyperactive and this I have taken from Patel et al general of neuroscience. So, you can see lots of signals seems to be hyperactive some of those phosphocaridin is however lower, but many of these seems to be altered. So, you can integrate these peaks find it out which the phosphorous containing energy currency becomes hyper metabolites in case of seizure brain and this was done using MRS. So, if you administer a drug and look at how the drug impact on the activity of the brain you again record a spectrum and look at the energy currency and the signals coming from that helps us what drug did. Although these were not detecting directly the drug metabolites, but we are looking at the response of that drug on the metabolism of these energy currency. So, that is what you can use the in view mode detection of the metabolites to understand the drug metabolism. Now, another example I am showing you for 1H MR spectroscopy for an A beta containing mice. So, A beta is a protein that aggregates in the Alzheimer's brain. So, the mice was basically induced with Alzheimer's and look at some of the signal that should come with a wild type and the Alzheimer's type of brain taken a section and did the MRS. And if you look at some of the signals that were different in the like a glutamate was same in a was same, but look at some of these seems to be different right seems to be different. So, what it shows that in case of Alzheimer's the metabolites that are present in the brain are different. This gives a very useful insight how actually disease changes the metabolic state of the brain and that essentially are used in understanding the metabolism in case altered metabolism in case of disease. So, here one can see it in vivo NMR spectrum of a cerebral cortex in the mice that are affected by the Alzheimer's. One can see the concentration of these seems to be lower here again it seems to be lower. So, differential concentration of like metabolites gives us an important insight of altered metabolism or in the right brain. Now, red was a smaller so you can put directly in the magnet for human it is little bit difficult, but that gives an idea how your drug regime or drug dosing should change. So, this study on animals will be very crucial in understanding how we can translate this idea into the human case. So, at the end I would just want to conclude in this week we started from understanding the drug design and then we went ahead and looked at how the NMR can guide us in having a better drug design, fragment based drug design, pharmacophore based drug design, various drug design and once the drug we have designed I just I wanted to look at whether the drug is we wanted to explore whether the drug is effective, how metabolism is impacted and how NMR can help in understanding the drug metabolism. So, slowly we went into the drug metabolism study where we looked at the in vitro part how we can use the body fluids to quantify the drug metabolism and today I just briefly touched upon how we can combine the in vivo approach for understanding the drug metabolism. This part of the NMR is called MRS magnetic resonance spectroscopy which helps in understanding the drug metabolism in vivo. This is a this is a field in itself you are if you are interested go and explore this. This is a beautiful field to understand the drug metabolism or metabolism in general without doing any surgery. So, just in vivo in non-invasive manner how you can use this beautiful concept of MRS for understanding what is happening in body. So, with this the next week we are transitioning into the another aspects of a structural biology in NMR called solid-district NMR and that is essentially whatever protein, whatever protein cannot be solubilized neither crystallizable that will be looked in using solid-district NMR. So, transitioning from the liquid state to solid state will be done next week and that is going to be last week for this course. So, looking forward to have you in the next week course which is solid-district NMR for biological molecule. So, thank you very much and looking forward to have some exciting questions from you and a vibrant engagement with you during the live session. Thank you very much.