 Okay students, good morning. So this week we are discussing how we can use NMR in drug discovery and last class I was discussing how we can use NMR in drug metabolism, understanding the drug metabolism. Why it is important to understand the drug metabolism as we discussed in the last lecture that the drug starts its journey from mouth and when it reaches to the cell where it has to act it remains only say 15 to 20 percent or very less because it has to pass through first past metabolism and this can vary from person to person because the enzymes that degrades the or metabolize these drugs cytochrome P450 family can be expressed differently in a different person. So in a healthy person it can have one dose or one effective concentration or we call it bio available concentration bioavailability and in diseased person or different person it can be different. Therefore, to understand the fate of the drug how it travels from the site of administration to the site of action is comes that comes under drug metabolism or pharmacokinetic and that is very important to understand to define a dosage because it can one dose cannot fit for all and that will not treat the patient effectively. So NMR plays a significant role in understanding in a quantitative manner this drug metabolism of pharmacokinetic how that we are going to do now, discuss now but last class we looked at that there are some enhancer that probably enhances the bioavailability. I showed you the example like a phthaloedocrine is taken like here if this drug is which is for blood pressure if it is taken with the fruit juice phthaloepine is taken with a grapefruit juice it increases the bioavailability you can see it here and that is what we concluded and it actually helps in having pronged effect. So plasma phthaloepine concentration also varies depending upon the cytochrome C450 CYP3A4 activity. So in patient with increased activity the bioavailability concentration in plasma is very less compared to healthy therefore understanding this drug metabolism in a quantitative manner is of paramount importance. So let us look at where NMR can help us right. So as you know NMR is non-invasive technique therefore it potterbs like minimally and can be used for drug metabolism. What we can take from body for understanding the drug metabolism any bio fluids which can be urine, the plasma, serum, tears, sweat anything that comes under bio fluid we can take our tissue extract or biopsy sample or even intact cell can be taken these are the in vitro approach we can do it like take out these and record the NMR spectrum in vitro condition or even one can do in vivo condition you can take whole animal model or a human subject that is a sister technique of NMR called MRI you can use these two techniques spectroscopy plus imaging to understand the drug metabolism in totality. So that is a in vivo use of NMR spectroscopy to understand the drug metabolism. And it has a unique ability because it permits both kind of study in vitro as well as in vivo. So like whole organism can be taken into the NMR magnet or MRI magnet and understand the drug metabolism and of course in vitro one can do with a bio fluids to tissue extract so and so forth. So what it allows one to do one can generate a metabolic signature of the drug right what kind of like what kind of metabolites comes out of a drug one can understand this it not only identify but it measures the metabolic flux like a when drug goes through different cycle of different stage of metabolism how the flux is changing how the like a flow of the drug is changing that can NMR can monitor as well as measure it quantitatively. It also many monitors the enzyme like some of the enzyme that we talked in the last class what is the activity of enzyme right. So once drug is administered enzymes comes into play how fast or how slow they are metabolizing you one can understand. So one can actually decipher the pathway or kinetic pathway of drug metabolism and one can even monitor what kind of modification that happens in the metabolic path when drug is administered or get metabolized does the does drug make any adduct or it combines with some other molecules when it gets metabolized. So essentially it can understand the effect of potter bands or toxins or any other things when drug goes from the site of administered to the circulation. So one can study all these or excretion one can study all of these. So typically the fluids that are used for doing the in vitro study like a diagnostic fluids plasma can be used serum can be used urine can be used saliva or any other secretion even tears can be used for understanding the enema like a drug metabolism using enema spectroscopy in a non-invasive manner. One of the prominent one is a urine why because whatever we take or consume as a drug finally it should be excreted and most of the things excreted out from urine right. So and like a taking urine is a minimal painful right everybody urinates easily you one can take urine identify the drug metabolism for like a taking plasma or serum to protect blood right. So not people or patients like it it is not a patient compliance body fluid. However urine comes in enough quantity and that can be used critically to understand how the drug metabolism happens. So everything that goes through kidney comes in urine and that can be used for detecting the drug metabolism right. So other than these body fluids if you want to do understand how drugs is reaching to different specialized location like a cerebral spinal location or thyroid or saliva whether it is sublingual or paratoid or multisubmaxillary or gastric location or even bile juices or pancreatic juices lots of these can be done with a localized enema spectroscopy or even immunotic fluid, follicular or in milk how drug is going into milk you know like a milk contamination is one of the major one. So like for babies mother's milk is very important. So generally one want to know that if what is the effect of drug on milk or even seminal vesicles how drug is effecting whether it is going or prosthetic many of such things can be understand by enema spectroscopy. The effect of a drug or the location of a drug metabolites can be identified by taking these body fluids and understanding using these drug metabolites in a quantitative manner. So what nuclei one can use for studying the drug metabolism. So see all the nuclei that we have studied can be exploited the prominent one is a hydrogen right it is a half spin and it is natural abundance right. So the chemical shift range is about 15 ppm this is actually everywhere present but there are some drawbacks with a hydrogen we will discuss that but you require a minimal concentration like in say 0.01 millimolar. The other one is deuterium then come lithium boron right carbon 13 carbon 13 can also be taken from the body fluids and can be exploited then it is N14, N15, O17 all these are NMR active nuclei like a fluorine is a it is a precieus nuclei that is used for understanding the drug metabolism. Similarly 31 p it is a natural abundance found in a many like if a nucleic acid the bones and all those it can be used. So these are some of the nuclei that can be exploited. So let us go little more detail and understand the pros and cons of each of these nuclei can be used for understanding the drug metabolism. So as you know proton is present in all drugs mostly in all drugs and it has a highest sensitivity. The natural abundance for protein proton is about 99.98. So it is a 100 percent natural abundance you can consider this. So this is the most prominent nuclei that can be exploited but there are some problem associated with a proton or hydrogen it is a it has a small chemical shift range of about 15 ppm and then because of the J coupling you see extensive multiplicity. So that actually makes the spectrum crowded and quantification of drug metabolites in those cases can be difficult right. So because of short chemical shift range and extensive multiplicity due to coupling homonuclear coupling makes spectral really crowded. So quantification or even the distinguishing the drug metabolites can be difficult. Another big problem that whatever body fluid you take the water is huge about 70 percent of all these will have will be dominated by water. Now you want to detect a very low concentration of drug from the body fluid where water is very high. Other than water there are proteins or lipids that are there. So before you start experiment you have to think about how we can get rid of proteins or lipid signal or reduce at least if you cannot illuminate. So there are techniques NMR based techniques that you saw for proteins like you can do water suppression, preset or gradient based gradient based actually suppression you can do it or to like concentrate more you can do freeze drying by using the T2 filters you can remove the signal of proteins or lipids. So these are some tricks that can be utilized for getting the signal of protons for protons from the drug metabolites in presence of huge amount of water, protein and lipids. So these are some of the tricks like some of the trick that can be applied and then one can detect the protons. Now not only in vitro even one can use proton in vivo but it comes with some problem and those are the problem because it is a poor nuclei for in vivo monitoring of drug because the tissue in homogeneity is there and in vivo you cannot spin sample or they are not fast mobile right. So this averaging of an isotropic interaction is not that prominent. Therefore lines are bound to be broad due to tissue in homogeneity or restricted molecular mobility because tissue cannot be like spin faster or all those and it can also be done because the tissue size can be like a little bigger than the whatever we have the homogeneous magnetic field. So magnetic field in homogeneity can come in this case because relatively large sample volume and use of lower magnetic field makes little bit difficult. You cannot put whole organism in a huge magnet of 20 Tesla, you have to restrict to 3 Tesla, 4 Tesla. In those case you do not want to put out this ecosystem of a organism by exposing to high magnetic field. So typically we do this experiment at a lower magnetic field and in those lower magnetic field the homogeneous space is limited. However our tissue or the whole organism that we are scanning can be bigger. So magnetic field in homogeneity is invariably there and that basically broadens the signal. So in vivo signal line width are substantially broader than what we obtained in vitro and those are the some of the limitation of using proton for in vivo drug metabolism experiment. The another important and beautiful nuclei is fluorine 19. So you know fluorine is interesting nuclei. First of all its nuclear spin is half has a relatively narrow line shape. It is a 100 percent natural abundance and very high sensitive like about 83 percent of proton. You can see it is a sensitive, it is a natural abundance is also high and gives sharp lines. You do not need to do anything special because it is not a quadrupole nuclei, it is a half integral nuclei, one spin is only half. So one can detect it. Other than this it has a large chemical shift range. So peaks cannot be crowded it is about 200 ppm line chemical shift. So peaks will be separated. So you can identify this and additional advantage which has it has a short longitudinal relaxation time. So you know if T1 is short that means you can afford to keep D1 also short. So experiment can be performed on a fast scale. The only requirement is you need to have a probe that can detect fluorine. Once you have the probe NMR probe where you can tune your RF to F19 you can do these experiments in a much cleaner manner and much beautiful manner. So if you have this one can do the rapid pulsing and you can increase the signal to noise ratio in per unit time in a quicker time. So and many drugs are actually fluorinated a large number of drugs are fluorinated that are in clinical use for those drugs NMR F19 NMR offers a powerful methods for monitoring their pharmacokinetic and metabolism. So F19 remember F19 is a beautiful nuclei to be used for understanding the drug metabolism and pharmacokinetic. Next is 13C right 13C you know for proteins it is widely used. Now in this case drugs cannot be isotopically labeled most of the time. So the only one one has to rely on 1.1 percent of natural abundance of the carbon 13 in any drug. But it has a large chemical shift. So basically actually if you can signal average to a large number of scan it can produce actually a reliable chemical structure for identifying the drug molecule right. Or one can isotopically label to enhance the signal to noise but that is typically cannot be done so easily because drugs cannot be isotopically labeled in a for all practical purpose. However for research purpose one can do it and then detect it. So mostly we have to rely on a natural abundance that is 1.1 percent right. So there are other tricks that one can apply. What are those tricks basically what we can do is like we detect on carbon 13 but we transfer the polarization from proton in a head core kind of experiment and then we can like do these kind of detection. So we can improve the sensitivity by doing this polarization transfer experiment and then you can use this drug like you can understand this drug metabolism using head core kind of experiment. Now 13c can be used in vitro as well as in vivo. And however actually it is of very limited use because of its sensitivity but yes it can be used. The another important nuclei for drug metabolism can be phosphorus 31. The good part is that it is a 100 percent natural abundance. It is a gyromagnetic ratio is about one-third of proton. So it is a significantly sensitive and phosphate is found in many drugs or even in the body. So one can look at the phosphorus signal. So indigenous phosphate and derivatives like a phospho mono esters or phosphodiester many of these can interfere from the signal of phosphorylated drugs and it metabolites. So one had to really identify what is coming from the drug and what is coming from our system. But yes it can be used and there are many phosphorus containing drugs not too many phosphorus containing drugs. So they are fairly rare but conceptually phosphorus 31 can also be used. Other than that there are some other nuclei which can probably use. Now lithium-based drugs are used for bipolar disorder or so. So lithium 7 can be used, boron 10 11 can be used, that deuterium, trisium, many of O17, N15, N14, platinum right. Platinum-based drugs are basically used in cancer treatment. So actually one can use platinum 195 for understanding the drug metabolism in case of cancerous tissue. So NMR offers a wide range of nuclei a wide range of possibility to be used for drug metabolism and few of the example we are going to understand. But before we delve into few of the example let us see what are the traditional techniques that are used for metabolic analysis. So these are low resolution I would say not low resolution. It is also high resolution techniques called HPLC high performance liquid chromatography, gas chromatography, the capillary electrophoresis, mass spectroscopy all of these are typically used. They require good part of them that they require minimum volume NMR requires a significant volume. However they may be getting perturbed with a column matrix or like depending upon the tension time or depending upon solvent use. So lots of possibility of modification that can come because of the nature of the experiment. The good part of NMR that you are not perturbing any sample. However it requires larger volume and it is less sensitive compared to these techniques HPLC, GCE, CEMS. But these are traditionally used in a quantitative manner to understand the pharmacokinetic use and these are like a widely used NMR has is picking up now. What are the problems of these traditional methods? They require separation. You have to separate that and then identify. So these are coupled methodology can be used. It requires optimization for separation. You cannot just take your sample and go and put in the HPLC and get your data. That is not the case for NMR. So here you need to optimize when your retention like looking at the retention time when your metabolite is coming. So some optimization is required. And many times you have to separate it like you have to separate a polar metabolites from non-polar metabolites smaller from bigger all these you have to do. So typically it takes time and these are slow techniques. And basically you have to do lots of like a lots of supervision. You require a high skill. It is a tedious. So manually intensive process all these are HPLC, CEMS, GCE, all these are like a manually intensive process. Now on the other hand NMR based drug metabolism study is a high throughput. You can use automatic sampler. Auto sampler put all the metabolites in one go 48, 24, whatever put it for overnight go and have a rest. Next day morning your results are there. The good part is the high resolution. Minimal effort you need for sample preparation. You just have to take your sample add D2O for locking and then you are ready to go. So you require really minimal sample preparation. High throughput, high resolution and less effort, less effort on sample preparation. And advantage is it is a quantitative. Looking at the peak intensity you can integrate those peak intensity and you can get the quantity of each of these metabolites that come out. Non-destructive you can use same sample for doing even LCMS, GCMS. After doing experiment you can take same sample and use orthogonal technique to understand whatever we are getting in NMR is correct or not. So you can be doubly sure using these samples. So it is a non-destructive same sample can be used and one metabolites can be detected simultaneously in a single experiment. So you do not need to separate it or isolate it. It can be used in a single experiment and it gives structural as well as quantitative information right. So what kind of a structure of metabolites is coming? Looking at the chemical shift pattern you can identify their structure and also looking at the intensity you can quantify and get the quantitative estimate of each of the metabolites. For few of the there is no significant for few of the nuclei there is no or little spectral interference coming from the indigenous molecule like F19 or 31p or lithium or carbon 13 you have a very minimal interference. However for protons we have interference as we discussed from coming from water, coming from protein, coming from lipid you can have an interference from these nucleons. So how you go about sample preparation? Suppose you are doing a experiment with one of the body fluid called plasma. So you have to take about 300 microlitre of plasma add buffer 300 microlitre you have 600 microlitre. So typically this is plasma and here is buffer where you have a H2O, TSP for internal reference the sodium azide for like so that bacterial growth does not happen and some buffer and then you need to have a D2O. So typically buffer concentration you can take some of these you do not need to centrifugation or shaking but because that can form bubble or foam you just do this take your experiment like your experimental setup is ready go and record the NMR. So you can see minimal sample preparation. If you are doing with a urine just take urine of about 90 percent and add D2O sodium azide TSP for internal reference sodium azide so that bacterial growth does not happen and you are all set to record experiment right. So again reemphasizing the fact that we are doing minimal sample preparation while recording the spectrum of these and you do not need very high magnet on a moderate magnet like a 500 mega hours, 600 mega 100 mega hours one can do experiment. Typically experiments are very easy like 1D no-G with pre-saturation of mixing time of 10 millisecond for urine you can do or even CPMG one can do with a CPMG delay of 200, 300 millisecond and echo time of 200 millisecond one can use it for plasma. Temperature typically you can have 298 room temperature and you do not need much scan just 32 like within a minutes experiment will be done and you can like a shear what this is because of water and coming from those so you have to get rid of water so you put like you suppress the water or put spectral region to 0 and you record few of the experiment like a 2D to have more dispersion like HSQC with some scans of 160 because you are you can get a better signal to noise in one go 1D or 2D whatever you record you are getting all the information from the body fluids. So typically what we get we get a spectral like this so suppose we are getting taking the human urine you have lots of spectrum like this looking at the database you can identify that these peaks are coming from glutamate, glutamine, butane or glycine right. So these are typically dominated now we know from healthy patient that how the signature comes from the healthy urine. Now if we are administrating drug and what extra peaks comes that you need to identify right. So let us take some of the example of how we can do that. So one healthy patient was given this drug called Fluor B Profane it is a anti-inflammatory drug the spectra were recorded on 600 megahertz. So you see you can exploit two nuclei here at least two nuclei one is proton that you can record the spectrum and you can find it out right. The other nuclei is beautifully located here fluorine it is a cleaner one if you are detecting on fluorine you have only signal coming from this drug and you can identify each metabolites its path that it travel. However for proton it will be little difficult because it is it is lots of indigenous signal will come from urine. So on 600 megahertz a sample of urine was collected after oral ingestion of 200 mg of this drug and then spectra were recorded right. So now the problem is lots of complication was coming. So one can do it one can actually couple the HPLC with NMR and you separate many of these whatever is coming depending upon their retention time and you can identify what happens after the drug is given. So what has happened that once drug was administered the drug made some kind of conjugate with the beta degluronic acid and that basically was also detected in the NMR spectrum. So depending upon like how the metabolites went what they found in the previous slide that two diastromeric form of beta degluronic acid conjugate of 40 hydroxy fluorobrufene was there and the resonance could be identified to resonance at 6.91 and 4.72 and other aromatic complex was coming at 7.19. So using this signature one can identify that where these are coming. So for this drug the resonances came at the 5.49 you can see somewhere here the signals were coming very high sorry not in this probably in this. So yeah somewhere here the signals were coming from the drug. So and the other signal come from the degluronic acid were located between 3 to 6 ppm. So one can identify all these resonances and this paper that I was referring they actually analyze all these and identify that how the drug level what adduct it made and the good part that this example emphasizes on that if we can couple HPLC with NMR one can identify all sorts of modification happens. So first you purify using HPLC and then go and record NMR and that gives wealth of information in this case. Another example that I want to show you is use of 13 CNMR for drug metabolic study. So these are the drugs so phenocetin and phenatidine. So basically these were administered and after one hour of administration basically the carbon 13 NMR was recorded from the healthy sample and also from hepatitis B suffering patient. You just look at the spectrum that is there and TSP was used for internal reference. You can see lots of modification is happening lots of extra peaks are coming and that helps us in identifying what is the fit or how the two person responds. So you can see the peaks differences that are coming here and additional peak that appeared at 66.9 ppm. So one can quantify it and look at the effect of this drug on a normal person on a person who is suffering from acute hepatitis. So these two example demonstrate the application of NMR in understanding the drug metabolism in a quantitative manner. And next class I will be discussing how we can use NMR spectroscopy for in vivo detection of drug metabolism. Till then I think it will be good to have questions from you. So looking forward for a healthy discussion over question and answer session. Thank you very much.