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See all Premium member Presentation Transcript DRUG DISCOVERY &DEVELOPMENT : DRUG DISCOVERY &DEVELOPMENT Principles and Procedures and Applications of instrumental methods Alliance Institute of Advanced Pharmaceutical & Health Sciences Prafulla Kumar Sahu M. Pharm (PhD.) www.allianceinstitute.org Slide 2: New drug development A process which applies to drugs, products and protocols to be used on human subjects. New Drug development is divided into 4 phases 1.Drug discovery 2.Pre clinical development 3.Clinical trial 4.Post approval 2 Alliance Organic Chemistry involved in Synthesis & Purification : Organic Chemistry involved in Synthesis & Purification Organic chemists synthesize new drug compounds as well as isolate and characterize natural products, such as alkaloids. In each case, we have to evaluate the complex relationships between chemical structure and pharmacological action. The pharmacological activity of a compound is an involved function of the structure, and very small changes may pro-foundly modify the pharmacological effect (SAR). Structural modifications: replacing one group with another at a specific point in the molecule, shifting the same group from place to place in the parent molecule, saturating valence bonds, modifying the acidity or basicity. Chromatographic techniques have been widely used for the purification of newly synthesized compounds. 3 Alliance Instrumental Techniques for Product Characterization : Instrumental Techniques for Product Characterization The first step in product characterization is to establish the precise chemical identity of the product. It is important to determine whether the material is a compound, i.e. a single chemical entity, a mixture of closely related compounds, mixture of isomers, or merely a loose molecular complex of readily dissociable components. Such information is fundamental to a proper evaluation of the biological properties of the material. 4 Alliance Product Characterization : Product Characterization For compounds of synthetic origin: Identity is usually clearly defined by the synthetic route employed. Modern spectroscopic techniques: 1H and 13C NMR IR (infrared spectroscopy) are sensitive tools for such purposes. 5 Alliance Physicochemical characterization techniques : Physicochemical characterization techniques Play a major role in the drug development process because they help us to understand the mechanism of drug delivery. Dissolution is the first step in drug delivery by solid oral dosage forms. but the determination of in vitro release profiles provides little information on the mechanism of the release of API from the product matrix. 6 Alliance Slide 7: It is required to assess (using instrumental techniques): Internal structure of the dosage form Microhomogeneity Morphology of the api in the dosage form instrumental techniques used: Microscopy and energy-dispersive x-ray spectroscopy X-ray powder diffraction, Thermal analysis, FTIR microspectroscopy, NMR imaging, Mass spectrometry, and Raman spectroscopy. The collection and interpretation of the results obtained from these techniques can be used to optimize formulation development and ensure the consistency, quality, and stability of solid dosage forms. 7 Alliance Product purification : Product purification Powerful separative techniques, particularly chromatography in all its many forms, provide sensitive methods. Liquid chromatography: is used for both purification and quantitative determination of the composition of mixtures of related compounds such as mixtures of isomers. Capillary gas chromatography: The speed and high separative power make it particularly useful, if highly specialized technique for the separation of complex mixtures during the research phase of drug development. 8 Alliance Slide 9: Where the product consists of more than one isomer, the isomers must be capable of separate identification and measurement to establish means of ensuring batch-to-batch consistency of isomer composition. For most optically active compounds, a simple polarimetric measurement will sufficient. Occasionally, however, where the measured rotation is small, measurements on a more sensitive instrument at other wavelengths either directly or after derivatisation may be necessary to secure adequate control of the product. In exceptional cases, the identification of optical isomers differing in only one of several chiral centers may call for the use of optical rotatory dispersion or circular dichroism to provide a degree of sensitivity that cannot be obtained from simple measurements of optical rotation. NMR is particularly valuable in distinguishing geometrical isomers. 9 Alliance Goals and objectives : Goals and objectives Clinical pharmacology and pharmacometrics Safety Activity Effectiveness Differentiation Successful FDA submission Market expansion and post marketing surveillance 10 Alliance Drug discovery process : Drug discovery process Screening consists of testing many compounds in assays relevant to the disease in question……HIT If the compound or its structural derivatives continue to show promise after further biological and chemical characters….LEAD 11 Alliance Slide 12: Two basic strategy are applied 1. Compound centered 2. Target centered 1. Compound centered approach: Natural products: isolated from plants, animals and microorganisms. ex. Morphine, Quinine, Atropine Adrenaline, Thyroxine Penicillin 12 Alliance Natural ligands : Natural ligands Examples: Dopamine- Parkinson- L-dopa Insulin – diabetes – exogenous insulin 13 Alliance Slide 14: 2. Target centered approach Use biochemical or molecular target Adv : pharmacological activity system assay 14 Alliance Structure based approach : Structure based approach Use 3-D structure of the target obtained through x-ray crystallography or NMR. Adv : -potency -limited number of drugs Disadv : -synthesis 15 Alliance Slide 16: Chemical synthesis: Based on SAR - ex. Histamine blockers Based on enantiomers - ex. dopa Rational approach: ex. Proton Pump Inhibitors. Molecular modelling: ex. COX 2 inhibitors Combinatorial chemistry: Biotechnology: ex. Growth factors, cytokines. 16 Alliance Lead optimization : Lead optimization In this stage where physical, chemical, biological and pharmacological properties are characterized and refined with the ultimate goal of selecting a single molecule to enter into clinical testing and formal drug development. Failure to demonstrate Efficacy Low bioavailability Extensive metabolism Low solubility Toxic effects Cost effectiveness in synthesis Reasons: 17 Alliance Drug discovery : Drug discovery Identifies interesting drug compounds, drug targets and delivery mechanisms with the potential for development into products. Characterize compounds and their targets Are “proof of concept” in nature Provide fundamental information on target biological system Identify lead compounds for further development 18 Alliance Pre clinical drug development : Pre clinical drug development Space the gap between drug discovery and clinical trials. Provide data on the safety and efficacy of the product Includes In vitro study, drug manufacturing, formulation and packaging, In vivo studies 19 Alliance In vitro study : In vitro study Intact cell lines assess drug effects on cells with specific DNA mutation Cell free system assay of enzyme activity, receptor binding, protein interaction with signal transduction 20 Alliance Drug manufacturing and formulation : Drug manufacturing and formulation Conducted under current good manufacturing practices (CGMP) guidelines IN-VIVO STUDIES PK study – ADME study PD study Therapeutic index Toxicological study 21 Alliance Toxicological study : Toxicological study Objectives : Acute study Method Aim : to provide safety/therapeutic index Observation 22 Alliance Sub acute study : Sub acute study aim : To identify the target organ To determine the clinical parameters To estimate the safety margin Method: Evaluation: Chronic study Similar to sub acute study except for the duration 23 Alliance Special study : Special study Effect on reproductive system and Teratogenicity Mutagenicity- ame’s test Carcinogenicity 24 Alliance Clinical drug evaluationAuthorization : Clinical drug evaluationAuthorization Investigational new drug (IND) submission -the rationale for the drug and patient group to be treated -all pre clinical safety and efficacy data -detailed plan for clinical development -CIB( clinical investigators brochure) Submitted to FDA for review and permission to proceed. 25 Alliance Slide 26: Is it safe? Does it work? Does it work in double blind trials? 26 Alliance APPROVAL : APPROVAL Once all clinical data has been submitted, reviewed and approval is granted to license in market Post marketing surveillance Approval takes 6 months to 2 years 27 Alliance Analytical Chemistry : Analytical Chemistry Definition: “The Science & art of determining composition of material in terms of element or compound content” Concerned with Chemical Characterization of mater Involves Qualitative and Quantitative determination 28 Alliance Slide 29: Goal of Analytical methods Qualitative analysis: A type of chemical analysis by which the analyte or analytes in a sample are identified. Quantitative analysis: A type of chemical analysis by which the amount of each analyte or analytes in a sample is determined. 29 Alliance Classification of Analytical Methods : Classification of Analytical Methods Analytical Techniques Chemical methods (Classical methods) Physical methods (Instrumental methods) 30 Alliance Chemical Method (Classical Method) : Chemical Method (Classical Method) Gravimetric Method Volumetric Method Acid Base titrations (Neutralization method) Oxidation reduction titrations (Redox method) Precipitation titrations Complexometric titrations 31 Alliance Classical Methods of Analysis : Classical Methods of Analysis Early years of chemistry Separation of analytes by precipitation, extraction, or distillation. Qualitative analysis by reaction of analytes with reagents that yielded products that could be recognized by their colors, boiling or melting points, solubilities, optical activities, or refractive indexes. Quantitative analysis by gravimetric or by titrimetric techniques. 32 Alliance Instrumental (Physical) Methods : Instrumental (Physical) Methods Instrumental Methods Optical methods Electrochemical methods Miscellaneous methods 33 Alliance Instrumental Methods : Instrumental Methods Measurement of physical properties of analytes - such as conductivity, electrode potential, light absorption or emission, mass-to-charge ratio, and fluorescence-began to be employed for quantitative analysis of inorganic, organic, and biochemical analytes Efficient chromatographic separation techniques are used for the separation of components of complex mixtures. Instrumental Methods of analysis (collective name for newer methods for separation and determination of chemical species.) 34 Alliance Optical Methods : Optical Methods Optical methods (Interaction of EMR) Absorption of EMR Emissions of EMR Scattering or Refraction of EMR Rotation of EMR UV-Visible IR NMR X-Ray Spectroscopy Fluoroscence Phosphorescence Flame Photometry Nephlometry Turbidometry Raman Spectroscopy Refractrometry Polarimetry 35 Alliance Electro Chemical Methods : Electro Chemical Methods pH metry & Potentiometry Conductometry Voltametry Polarography Amperometery Coulometry 36 Alliance Miscellaneous methods : Miscellaneous methods Thermometric Analysis: DSC Thermo Gravimetric Analysis 37 Alliance Slide 38: Classification of Analytical Methods 1. Classical methods Qualitative – identification by color, indicators, boiling or melting points, odors Quantitative – mass or volume (e.g. gravimetric, volumetric) Based on goal of application: 38 Alliance Slide 39: 2. Instrumental methods Qualitative – chromatography, electrophoresis and identification by measuring physical property (e.g. spectroscopy, electrode potential) Quantitative – measuring property and determining relationship to concentration (e.g. spectrophotometry, mass spectrometry) Often, same instrumental method used for Qualitative and Quantitative analysis. 39 Alliance Slide 40: Types of Instrumental Methods Radiation emission Emission spectroscopy (X-ray, UV, visible), fluorescence, phosphorescence, luminescence Radiation absorption Absorption spectroscopy spectrophotometry, photometry, NMR electron spin resonance 40 Alliance Slide 41: Signal Example Method Radiation Scattering Raman spectroscopy Radiation refraction Refractometry Radiation diffraction X-ray and Electron diffraction method Radiation rotation Polarimetry Electrical potential Potentiometry 41 Alliance Slide 42: Signal Example Method Electrical charge Coulometry Electrical current Voltammetry – amperometry polarography Electrical resistance Conductometry Mass Gravimetry Mass-to-charge ratio Mass spectrometry Rate of reaction Flow injection analysis 42 Alliance Slide 43: Chemical and physical properties employed in instrumental methods: 43 Alliance Slide 44: Instruments for Analysis Example: Spectrophotometry Instrument: spectrophotometer Stimulus: monochromatic light energy Analytical response: photocell Data: electrical current Data processor: current meter Readout: meter scale Encoded information 44 Alliance Slide 45: Encoded information: detector : device that indicates changes in environment transducer : device that converts non-electrical to electrical data Non-electrical domains Electrical domains Physical Current (light intensity, colour) Chemical (pH) Voltage 45 Alliance Advantages of Instrumental Methods : Advantages of Instrumental Methods High Sensitivity Accuracy Small Sample can be used Measurements obtained are reliable & Reproducible Fast Determination Complex sample can be handled easily Process can be made automatic 46 Alliance Limitations of Instrumental methods : Limitations of Instrumental methods High Cost Necessary to use reference substance Skilled Persons are required 47 Alliance Slide 48: Selecting an Analytical Method Based on answers to the following questions: What accuracy and precision are required? How much sample is available? What is the concentration range of the analyte? What components of the sample will cause interference? What are the physical and chemical properties of the sample matrix? How many samples are to be analyzed? 48 Alliance IR Spectroscopy : IR Spectroscopy INFRARED REGION : INFRARED REGION 4000cm-1 to 600 cm-1 IR spectrum: Plot of Trasmittance/Absorbance VS Frequency of radiation SAMPLE IR TRANSMITTED ABSORBED 50 Alliance Molecular vibration : Molecular vibration Atoms joined by covalent bonds undergo continual vibrations relative to each other The energies associated with these vibrations are quantitized. Within a molecule only specific vibrational energy levels are allowed. The energies associated with transitions between vibrational energy levels for most covalent bonds are from 2 to 10 kcal/mol (8.4 to 42kj/mol) 51 Alliance Infrared spectroscopy : Infrared spectroscopy Vibrational IR spectral region covers 2.5 x 10-6 (2.5 µm) to 2.5 x 10-5 (25 µm) Absorption of IR radiation in this region causes bond to change from a lower vibrational energy level to a higher one IR SPECTRUM REPRESENTS CHANGES IN ENERGY BROUGHT BY TRANSITION OF MOLECULES FROM ONE VIBRATIONAL OR ROTATIONAL ENERGY STATE TO ANOTHER. 52 Alliance Slide 53: IR causes a vibration only if there is a change in dipole during vibration Therefore symmetric bonds are inactive. CH3 - CH3 will not observe IR stretch for C-C bond of ethane 53 Alliance Infrared (IR) and Raman Spectroscopies : Infrared (IR) and Raman Spectroscopies Infrared (IR) and Raman spectroscopies have been standard methods of analytical pharmacy and chemistry for a long time. IR and Raman spectra, which are complementary to each other, provide images of vibrations of the atoms of a compound. Therefore, both techniques are also referred to as vibrational spectroscopy. An IR spectrum is obtained by passing infrared radiation through a sample and determining what fraction of the incident radiation is absorbed at particular frequency. On the other hand, a Raman spectrum is obtained by focusing monochromatic radiation on a sample and analyzing the scattered light as a function of frequency. IR spectroscopy is based on the absorption of electromagnetic radiation by a molecular system, Raman spectroscopy relies upon inelastic scattering of electromagnetic radiation by a molecular system. 54 Alliance Applications of IR : Applications of IR Fourier Transformed Infrared (IR) and Raman Spectroscopy are important tools for the solid state characterization of pharmaceutical solids and for the identification of their chemical structures. Generally both methods are applied in combination with other methods for solid state characterization of pharmaceutical solids (e.g. X- ray powder diffraction, DSC, TG). characterization of polymorphism and detection in drug product. Both methods are also suitable for the identification/ detection of the polymorphic form in the tablet. 55 Alliance Applications of FTIR : Applications of FTIR Very high resolution required- Gaseous Mixtures Study of samples having very high absorption Study of samples with weak absorption bands Used in protein structure determination Used in characterizing Adrenocarcinoma Very small sample size: Obtaining Reflection spectra IR emission study 56 Alliance Applications of Near IR : Applications of Near IR NIR: good penetration properties Minimal sample penetration required Thick layers can be analyzed Not much useful for identification Quantitative analysis of compounds containing functional groups made of H bonded to O, C, N Determination of 10, 20, 30 amines 57 Alliance Applications of ATR-FTIR : Applications of ATR-FTIR Surface analysis of biological structures Monitoring reaction processes Evaluation of fermented foods 58 Alliance Pharmaceutical applications of Mid-IR and Raman spectroscopy : Pharmaceutical applications of Mid-IR and Raman spectroscopy Mid-IR and Raman spectroscopy are versatile tools in pharmaceutics and biopharmaceutics, with a wide field of applications ranging from characterization of drug formulations to elucidation of kinetic processes in drug delivery. New developments in applications of these methods for studying drug delivery systems. FTIR-ATR is a well-established standard method used to study drug release in semisolid formulations, drug penetration, and influence of penetration modifiers; it is also capable of in vivo studies. 59 Alliance Slide 60: FTIR-PAS (photo acoustic spectroscopy) has been applied to: Measure drug content in semisolid and solid formulations, Determine drug penetration into artificial and biological membranes. Raman spectroscopy can be used to: Characterize the structure of colloidal drug carrier systems. In vivo studies. Recently, there has been tremendous technical improvement in vibrational microspectroscopy: FTIR imaging shows great promise in its ability to visualize the drug and excipient distribution in pharmaceutical formulations such as tablets and therapeutic transdermal systems, as well as to reveal the mechanism of drug release. Furthermore, this unique technique offers completely new possibilities to study the lateral diffusion of drugs. 60 Alliance Slide 61: NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY -an ideal tool for Diagnosis & Drug Design 61 Alliance INTRODUCTION : INTRODUCTION BASIC PRINCIPLE IN NMR Atomic Nuclei – tiny magnets In external Magnetic Field - allign or oppose If irradiated with EMR of proper frequency – Resonance √ α βo PARAMETERS Chemical shifts Spin lattice relaxation time 62 Alliance Slide 63: NUCLEAR SPIN STATES - HYDROGEN NUCLEUS + 1/2 - 1/2 The two states are equivalent in energy in the absence of a magnetic or an electric field. + + The spin of the positively charged nucleus generates a magnetic moment vector, m. m m TWO SPIN STATES 63 Alliance Slide 64: 64 Alliance Slide 65: RESONANCE Absorption of energy by the spinning nucleus 65 Alliance Resonance : Resonance Resonance: Matching of natural frequency with the applied frequency. In NMR the applied radio frequency has to be match with the precessional frequency of nuclei (which is the consequences of applied external magnetic field) Absorption of radio frequency occur by nuclei after resonance is achieved. 66 Alliance Slide 67: Precessional frequency (n) The number of revolution per second made by magnetic moment vector of the nucleus around the external magnetic field B0 OR Precessional frequency is equal to the frequency of electromagnetic radiation in MHz (Mega cycles/sec) necessary to induce a transition from one spin state to other. 67 Alliance Nuclear Spin Energy Levels : Nuclear Spin Energy Levels Bo +1/2 -1/2 In a strong magnetic field (Bo) the two spin states differ in energy. aligned unaligned N S 68 Alliance Absorption of Energy : Absorption of Energy Bo +1/2 -1/2 +1/2 -1/2 DE = hn DE quantized Radiofrequency Applied Field Aligned Opposed 69 Alliance Slide 70: Bo DE + 1/2 - 1/2 = kBo = hn degenerate at Bo = 0 increasing magnetic field strength THE ENERGY SEPARATION DEPENDS ON Bo 70 Alliance The Larmor Equation : The Larmor Equation g n = 2p Bo g is a constant which is different for each atomic nucleus (H, C, N, etc) DE = kBo = hn can be transformed into gyromagnetic ratio g strength of the magnetic field frequency of the incoming radiation that will cause a transition n α Bo Stronger magnetic fields (Bo) cause the instrument to operate at higher frequencies (n). 71 Alliance NMR Signals : NMR Signals The number of signals shows how many different kinds of protons are present. The location of the signals shows how shielded or deshielded the proton is. The intensity of the signal shows the number of protons of that type. Signal splitting shows the number of protons on adjacent atoms. 72 Alliance DIFFERENT TECHNIQUES : DIFFERENT TECHNIQUES Based on nuclei H1 NMR C13 NMR F13 NMR & P31 NMR N15 NMR & O17 NMR CWNMR (continuous wave NMR) FTNMR 2D_NMR &3D_NMR COSY _CORRELATION SPECTROSCOPY MRI_MAGNETIC RESONANCE IMAGING ESR_ELECTRON SPIN RESONANCE SPECTROSCOPY 73 Alliance APPLICATIONS OF NMR IN MEDICINE : APPLICATIONS OF NMR IN MEDICINE CLINICAL APPLICATION OF PROTON IMAGING IN DIAGNOSIS BRAIN Distinguishing gray matter & white matter Imaging posterior fossae, brain stem, spinal cord Detect demyelinating lesions, tumors, hemorrhages, infarctions Slide 75: ABDOMEN Metabolic liver disease Measures liver iron over load in hemochromatosis Focal areas of inflammation in chronic active hepatisis KIDNEYS Distinguishing renal cortex & medulla To evaluate transplanted kidney PELVIS Differentiates between Benign prostatic hyperplasia & prostatic carcinoma Detects bladder tumours 75 Alliance Slide 76: LUNG Assessing abnormalities Respiratory gating HEART Tomographic images of heart muscle, chambers, valvular structures ECHO-PLANAR TECHNIQUE Discrimination between infarcted, ischemic & normal myocardium BREAST 3D-NMR & single-slice planar imaging in detecting breast abnormalities 76 Alliance : MUSCULO SKELETAL SYSTEM Demonstrates Osteo myelitis, tumor metastasis in vertebral bodies & pelvic bones Images of muscles, tendons, ligaments BLOOD VESSELS & FLOW Atherosclerotic vascular disease Assess blood flow in major vessels. INVIVO SPECTROSCOPY Chemical shift phenomenon Diagnosis of rare disease to inborn errors- Mc Ardle’s syndrome INVIVO Analysis of Bone flouride content. NMR IMAGING STUDIES : NMR IMAGING STUDIES Eliminates risk of x-radiation Excellent spatial & contrast resolution Detecting diseases at earlier stages 78 Alliance NMR IN PHARMACEUTICAL RESEARCH : NMR IN PHARMACEUTICAL RESEARCH Leading technology for 3-D structure determination of bio-macromolecules Studying protein structure SAR by NMR – Novel lead compounds Chemical shift mapping – Structural information on the binding modes and site positions Molecular dynamics, conformational analysis SHAPES – Lead generation NMR – SOLVE 79 Alliance Slide 80: Structural genomics Identifying gene products in disease Targets of drug design. Development of Novel drug delivery systems Enhancing HTS assays Thousands of compounds Cryogenic NMR technology / cryoprobes 80 Alliance Slide 81: 1.Identification of structural isomers 2.Detection of hydrogen bonding 3.Detection of aromaticity 4.Distinction between cis and trans isomer 5.Detection of electronegative atom/group 6.Detection of some double bond character due to resonance 7.Importance in quantitative analysis 81 Alliance NMR Nobel Prize Laureates : NMR Nobel Prize Laureates The Nobel Prize in Physics 1943, Ohostern, USA – Discovery of magnetic moment of proton The Nobel Prize in Physics 1952- IsidorJ. Rafi,USA – Discovery of new methods for nuclear magnetic precision The Nobel Prize in Chemistry 1991 – Felix Bloch, USA and Edward M. Purcell, USA – Methodology of high resolution NMR. The Nobel Prize in Chemistry 2002 – Kurt Wuthrich, Switzerland – Determining 3-D Structure by NMR. The Nobel Prize in Medicine 2003 – Paul C. Lauterbur, USA and Peter Mans field, U.K. – Discoveries concerning MRI. 82 Alliance MASS SPECTROMETRY : MASS SPECTROMETRY 83 Alliance Introduction : Introduction Mass spectrometry is employed to analyze combinatorial libraries, sequence biomolecules, Structure elucidation of unknowns, Environmental and forensic analytics, Quality control of drugs, flavors and polymers 84 Alliance Slide 85: The basic principle of mass spectrometry (MS): To generate ions from either inorganic or organic compounds by any suitable method, To separate these ions by their mass-to-charge ratio (m/z) And to detect them qualitatively and quantitatively by their respective m/z and abundance. Besides electrons, (atomic) ions or photons, energetic neutral atoms and heavy cluster ions can also be used to effect ionization of the analyte. The time-of-flight (TOF) analyzer, ion separation by m/z can be effected in field free regions, too, provided the ions possess a well defined kinetic energy at the entrance of the flight path. The analyte may be ionized thermally, by electric fields or by impacting energetic electrons, ions or photons. 85 Alliance Application of LDI (Laser Desorption/Ionization) : Application of LDI (Laser Desorption/Ionization) LDI of peptides and oligosaccharides LDI is much better suited for the analysis of organic and inorganic salts Molecules with large conjugated π-electron systems Organic dyes as contained in ball point inks Porphyrins UV light-absorbing synthetic polymers LDI presents a useful alternative to MALDI in the low-mass range. In addition, solvent free sample preparation can be employed with insoluble analytes. However, LDI is a “harder” method than MALDI and fragmentation 86 Alliance Applications of MALDI : Applications of MALDI MALDI-MS of Synthetic Polymers: Fingerprints by MALDI-MS Carbohydrates by MALDI-MS Structure Elucidation of Carbohydrates by MALDI Oligonucleotides in MALDI 87 Alliance Slide 88: MALDI is the method of choice for the analysis of synthetic polymers because it usually provides solely intact and singly charged quasimolecular ions over an essentially unlimited mass range. While polar polymers such as poly (methylmethacrylate) (PMMA), polyethylene glycol (PEG), and others readily form [M+H]+ or [M+alkali]+ ions, Nonpolar polymers like polystyrene (PS) non-functionalized polymers like polyethylene (PE) can only be cationized by transition metal ions in their 1+ oxidation state. The formation of evenly spaced oligomer ion series can also be employed to establish an internal mass calibration of a spectrum. The most important parameters that can be determined by MALDI are number-average molecular weight (Mn) weight-average molecular weight (Mw) molecular weight distribution expressed as polydispersity (PD) 88 Alliance Application of Electrospray Ionization : Application of Electrospray Ionization ESI of Small Molecules (polar analytes) Peptide and protein characterization including their complete sequencing ESI is applied to Metal Complexes (ionic) and related compounds ESI of Surfactants: Cationic, anionic and non-ionic surfactants are readily detected by ESI Oligonucleotides, DNA, and RNA are best analyzed by negative-ion ESI ESI of Oligosaccharides and closely related compounds such as glycoproteins, gangliosides, liposaccharides etc. permits molecular weight determination and structure elucidation 89 Alliance LC-MS Applications in drug development : LC-MS Applications in drug development 90 Alliance LC-MS Applications in drug development : LC-MS Applications in drug development 91 Alliance LC-MS Applications in Drug Discovery : LC-MS Applications in Drug Discovery 92 Alliance Peptide Mapping : Peptide Mapping Peptide Mapping: The identification of proteins is essential for understanding biological process and the function of the protein during normal and disease states. The resulting insights lead to the development of therapies for intervention, and ultimately, the cure of disease. The information and knowledge derived from this type of study are extremely valuable for activities involved with target identification activities during drug discovery. Two-dimensional gel electrophoresis (2-DGE) is the primary analysis tool used in the pharmaceutical industry to characterize the expression of proteins. Mass spectrometry-based methods are emerging as an important partner with 2-DGE for correlating proteins to their function. 93 Alliance Peptide Mapping : Identification schemes that involve LC/MS and MALDI-TOF techniques have been applied to a broad range of events that involve proteins such as covalent modification, proteolytic cleavage, and binding. Usually, the focus of LC/MS methods for protein characterization is on four distinct goals: (1) confirmation of putative sequence; (2) identification of amino acid modifications; (3) identification of known proteins; (4) sequence determination of unknown proteins. 94 Peptide Mapping Alliance Glycoprotein Mapping : Glycoprotein Mapping Similar analysis strategies can be applied for the peptide mapping of glycoproteins combined in-source collisionally induced dissociation (CID) with LC/MS/MS to identify sites of N- and O-linked glycosylation. This novel approach uses a series of LC/MS and LC/MS/MS experiments to generate peptide maps and to selectively screen for glycoproteins. The method is based on the characteristic fragmentation of glycoproteins to form a N-acetylhexosamine (HexNAc+) fragment ion at m/z 204. This fragment ion serves as a diagnostic marker for N- and O-linked glycopeptides. 95 Alliance Slide 96: A mixture of peptides and glycopeptides is generated when the glycoprotein is reduced, alkylated, and enzymatically digested. A series of three separate experiments, is used to identify the N- and O-linked glycopeptides. The first experiment uses LC/MS to provide a map of the peptide portion of the protein and to indicate the presence of glycopeptides. The second experiment involves the use of LC/MS/MS to screen the mixture for compounds that fragment to yield the diagnostic m/z 204 (HexNAc+). N- and O-linked glycopeptides are both identified. A final experiment is performed on a sample treated with peptide N:glycosidase F (PNGase F) to release the N-linked oligosaccharides and to identify the O-linked glycopeptides exclusively. 96 Alliance Slide 97: 97 Alliance Slide 98: Additional structural analysis of glycoproteins generally requires fragmentation by chemical or enzymatic cleavage and by separation or isolation. On-line LC/MS methods provide an integrated approach for this type of analysis. 98 Alliance Natural Products Dereplication : Natural Products Dereplication Natural products offer a source of unique chemical diversity for the pharmaceutical industry. Drugs derived from natural products have been introduced for the treatment of cancer, immunosuppression, cardiovascular therapy, and anti-infective therapy. Most antibiotics come from secondary metabolites of soil microorganisms that inhibit bacteria or fungi. Large scale screening of microorganism fermentations followed by isolation and structure elucidation is required. Because many natural products have been previously identified, approaches that avoid time-consuming isolation and provide quick elucidation are essential. 99 Alliance Slide 100: a new strategy was introduced by Ackermann et al. that uses an on-line ESI±LC/MS approach that integrates multi-component identification, fraction collection, and sample preparation for bioactivity screening (1996). Using this LC/MS based approach, crude extracts are screened without extensive purification and chemical analysis. Less material is required due to the sensitivity of the technique and chromatographic resolution is retained. This resolution would ordinarily be relatively poor with a fraction collection process. Furthermore, because the ESI acts as a concentration- dependent detector, the HPLC effluent can be split between mass spectrometry analysis and fraction collection. Thus, molecular weight information is obtained, and approximately 90% of the fractionated material is recovered for biological testing. 100 Alliance Slide 101: 101 Alliance Bioaffinity Screening : Bioaffinity Screening Combinatorial chemistry has changed the strategy of drug candidate synthesis. As a result, hundreds of thousands of compounds are now screened against a particular biological target. Once activity is determined for a mixture, the identification of the active component(s) is necessary. However, this approach requires a considerable amount of resources. To reduce the time and resources required for screening the large number of compounds produced by combinatorial chemistry, approaches featuring the parallel screening of mixtures of compounds (20±30) have been investigated. The recent studies performed by Anderegg and coworkers describe the use of bioaffinity selection LC/MS methods for the identification of active mixture component(s) (Davis et al., in press). This approach features an integrated bioaffinity based LC/MS screening method to separate and identify Lead compounds from mixtures. 102 Alliance Slide 103: 103 Alliance In Vivo Drug Screening : In Vivo Drug Screening Technology for automated highthroughput bioanalysis. focused on methods for determining multicomponent mixtures of drug candidates that involve pharmacokinetics and metabolic stability. These activities were performed during the preclinical and clinical stages of drug development. The need to provide an early assessment of these properties resulted in new strategies for in vivo drug screening during the drug discovery stage. LC/MS/MS approaches, using relatively simple isolation and chromatography conditions, present an effective approach to evaluate new lead compounds for subsequent development or selection of an optimal drug candidate within a specific therapeutic area or a targeted class of compounds. 104 Alliance Metabolic Stability Screening : Metabolic Stability Screening Recently, the use of fast gradient elution LC/MS techniques was described for high throughput metabolic stability screening (Ackermann et al., 1998). The method uses a HPLC column-switching apparatus to desalt and analyze lead candidates incubated with human liver microsomes. The rapid structure identification of metabolites using LC/MS/MS with an ion trap mass spectrometer (Lopez et al., 1998). In this study, metabolic stability analysis is automated to provide maximum structural information in combination with predictive strategies for biotransformation. 105 Alliance Slide 106: This feature is unique and avoids the multiple (2±4) injections that are necessary with other MS/MS configurations (e.g., tandem quadrupole). Along with the significant savings in time, detailed structure information is generated, which enables a comprehensive analysis of substructure relationship to be constructed for each metabolite. These automated studies provide unique advantages during drug discovery, and provide an early perspective on the metabolically labile sites, or ``soft spots'' of a drug candidate. This knowledge is useful during lead optimization activities, and can lead to the initiation of proactive research efforts that deal with metabolism-guided structural modification and toxicity. 106 Alliance Applications of LC/MS in Preclinical Development. : Applications of LC/MS in Preclinical Development. 107 Alliance Application of LC/MS in Clinical Development : Application of LC/MS in Clinical Development 108 Alliance Applications of LC/MS in Manufacturing : Applications of LC/MS in Manufacturing 109 Alliance Applications of LC-MS : Applications of LC-MS Toxicological studies, finding drugs and toxins in variety of materials with smaller sample volume Urine analysis for toxicology Dope test: doping agents and drugs of abuse (steroids, diuretics) Impurity profiling Metabolite studies Bio-equivalence and Bioavailability studies Molecular weight Assay of drugs, intermediates etc. 110 Alliance You do not have the permission to view this presentation. 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Pharm (PhD.) www.allianceinstitute.org Slide 2: New drug development A process which applies to drugs, products and protocols to be used on human subjects. New Drug development is divided into 4 phases 1.Drug discovery 2.Pre clinical development 3.Clinical trial 4.Post approval 2 Alliance Organic Chemistry involved in Synthesis & Purification : Organic Chemistry involved in Synthesis & Purification Organic chemists synthesize new drug compounds as well as isolate and characterize natural products, such as alkaloids. In each case, we have to evaluate the complex relationships between chemical structure and pharmacological action. The pharmacological activity of a compound is an involved function of the structure, and very small changes may pro-foundly modify the pharmacological effect (SAR). Structural modifications: replacing one group with another at a specific point in the molecule, shifting the same group from place to place in the parent molecule, saturating valence bonds, modifying the acidity or basicity. Chromatographic techniques have been widely used for the purification of newly synthesized compounds. 3 Alliance Instrumental Techniques for Product Characterization : Instrumental Techniques for Product Characterization The first step in product characterization is to establish the precise chemical identity of the product. It is important to determine whether the material is a compound, i.e. a single chemical entity, a mixture of closely related compounds, mixture of isomers, or merely a loose molecular complex of readily dissociable components. Such information is fundamental to a proper evaluation of the biological properties of the material. 4 Alliance Product Characterization : Product Characterization For compounds of synthetic origin: Identity is usually clearly defined by the synthetic route employed. Modern spectroscopic techniques: 1H and 13C NMR IR (infrared spectroscopy) are sensitive tools for such purposes. 5 Alliance Physicochemical characterization techniques : Physicochemical characterization techniques Play a major role in the drug development process because they help us to understand the mechanism of drug delivery. Dissolution is the first step in drug delivery by solid oral dosage forms. but the determination of in vitro release profiles provides little information on the mechanism of the release of API from the product matrix. 6 Alliance Slide 7: It is required to assess (using instrumental techniques): Internal structure of the dosage form Microhomogeneity Morphology of the api in the dosage form instrumental techniques used: Microscopy and energy-dispersive x-ray spectroscopy X-ray powder diffraction, Thermal analysis, FTIR microspectroscopy, NMR imaging, Mass spectrometry, and Raman spectroscopy. The collection and interpretation of the results obtained from these techniques can be used to optimize formulation development and ensure the consistency, quality, and stability of solid dosage forms. 7 Alliance Product purification : Product purification Powerful separative techniques, particularly chromatography in all its many forms, provide sensitive methods. Liquid chromatography: is used for both purification and quantitative determination of the composition of mixtures of related compounds such as mixtures of isomers. Capillary gas chromatography: The speed and high separative power make it particularly useful, if highly specialized technique for the separation of complex mixtures during the research phase of drug development. 8 Alliance Slide 9: Where the product consists of more than one isomer, the isomers must be capable of separate identification and measurement to establish means of ensuring batch-to-batch consistency of isomer composition. For most optically active compounds, a simple polarimetric measurement will sufficient. Occasionally, however, where the measured rotation is small, measurements on a more sensitive instrument at other wavelengths either directly or after derivatisation may be necessary to secure adequate control of the product. In exceptional cases, the identification of optical isomers differing in only one of several chiral centers may call for the use of optical rotatory dispersion or circular dichroism to provide a degree of sensitivity that cannot be obtained from simple measurements of optical rotation. NMR is particularly valuable in distinguishing geometrical isomers. 9 Alliance Goals and objectives : Goals and objectives Clinical pharmacology and pharmacometrics Safety Activity Effectiveness Differentiation Successful FDA submission Market expansion and post marketing surveillance 10 Alliance Drug discovery process : Drug discovery process Screening consists of testing many compounds in assays relevant to the disease in question……HIT If the compound or its structural derivatives continue to show promise after further biological and chemical characters….LEAD 11 Alliance Slide 12: Two basic strategy are applied 1. Compound centered 2. Target centered 1. Compound centered approach: Natural products: isolated from plants, animals and microorganisms. ex. Morphine, Quinine, Atropine Adrenaline, Thyroxine Penicillin 12 Alliance Natural ligands : Natural ligands Examples: Dopamine- Parkinson- L-dopa Insulin – diabetes – exogenous insulin 13 Alliance Slide 14: 2. Target centered approach Use biochemical or molecular target Adv : pharmacological activity system assay 14 Alliance Structure based approach : Structure based approach Use 3-D structure of the target obtained through x-ray crystallography or NMR. Adv : -potency -limited number of drugs Disadv : -synthesis 15 Alliance Slide 16: Chemical synthesis: Based on SAR - ex. Histamine blockers Based on enantiomers - ex. dopa Rational approach: ex. Proton Pump Inhibitors. Molecular modelling: ex. COX 2 inhibitors Combinatorial chemistry: Biotechnology: ex. Growth factors, cytokines. 16 Alliance Lead optimization : Lead optimization In this stage where physical, chemical, biological and pharmacological properties are characterized and refined with the ultimate goal of selecting a single molecule to enter into clinical testing and formal drug development. Failure to demonstrate Efficacy Low bioavailability Extensive metabolism Low solubility Toxic effects Cost effectiveness in synthesis Reasons: 17 Alliance Drug discovery : Drug discovery Identifies interesting drug compounds, drug targets and delivery mechanisms with the potential for development into products. Characterize compounds and their targets Are “proof of concept” in nature Provide fundamental information on target biological system Identify lead compounds for further development 18 Alliance Pre clinical drug development : Pre clinical drug development Space the gap between drug discovery and clinical trials. Provide data on the safety and efficacy of the product Includes In vitro study, drug manufacturing, formulation and packaging, In vivo studies 19 Alliance In vitro study : In vitro study Intact cell lines assess drug effects on cells with specific DNA mutation Cell free system assay of enzyme activity, receptor binding, protein interaction with signal transduction 20 Alliance Drug manufacturing and formulation : Drug manufacturing and formulation Conducted under current good manufacturing practices (CGMP) guidelines IN-VIVO STUDIES PK study – ADME study PD study Therapeutic index Toxicological study 21 Alliance Toxicological study : Toxicological study Objectives : Acute study Method Aim : to provide safety/therapeutic index Observation 22 Alliance Sub acute study : Sub acute study aim : To identify the target organ To determine the clinical parameters To estimate the safety margin Method: Evaluation: Chronic study Similar to sub acute study except for the duration 23 Alliance Special study : Special study Effect on reproductive system and Teratogenicity Mutagenicity- ame’s test Carcinogenicity 24 Alliance Clinical drug evaluationAuthorization : Clinical drug evaluationAuthorization Investigational new drug (IND) submission -the rationale for the drug and patient group to be treated -all pre clinical safety and efficacy data -detailed plan for clinical development -CIB( clinical investigators brochure) Submitted to FDA for review and permission to proceed. 25 Alliance Slide 26: Is it safe? Does it work? Does it work in double blind trials? 26 Alliance APPROVAL : APPROVAL Once all clinical data has been submitted, reviewed and approval is granted to license in market Post marketing surveillance Approval takes 6 months to 2 years 27 Alliance Analytical Chemistry : Analytical Chemistry Definition: “The Science & art of determining composition of material in terms of element or compound content” Concerned with Chemical Characterization of mater Involves Qualitative and Quantitative determination 28 Alliance Slide 29: Goal of Analytical methods Qualitative analysis: A type of chemical analysis by which the analyte or analytes in a sample are identified. Quantitative analysis: A type of chemical analysis by which the amount of each analyte or analytes in a sample is determined. 29 Alliance Classification of Analytical Methods : Classification of Analytical Methods Analytical Techniques Chemical methods (Classical methods) Physical methods (Instrumental methods) 30 Alliance Chemical Method (Classical Method) : Chemical Method (Classical Method) Gravimetric Method Volumetric Method Acid Base titrations (Neutralization method) Oxidation reduction titrations (Redox method) Precipitation titrations Complexometric titrations 31 Alliance Classical Methods of Analysis : Classical Methods of Analysis Early years of chemistry Separation of analytes by precipitation, extraction, or distillation. Qualitative analysis by reaction of analytes with reagents that yielded products that could be recognized by their colors, boiling or melting points, solubilities, optical activities, or refractive indexes. Quantitative analysis by gravimetric or by titrimetric techniques. 32 Alliance Instrumental (Physical) Methods : Instrumental (Physical) Methods Instrumental Methods Optical methods Electrochemical methods Miscellaneous methods 33 Alliance Instrumental Methods : Instrumental Methods Measurement of physical properties of analytes - such as conductivity, electrode potential, light absorption or emission, mass-to-charge ratio, and fluorescence-began to be employed for quantitative analysis of inorganic, organic, and biochemical analytes Efficient chromatographic separation techniques are used for the separation of components of complex mixtures. Instrumental Methods of analysis (collective name for newer methods for separation and determination of chemical species.) 34 Alliance Optical Methods : Optical Methods Optical methods (Interaction of EMR) Absorption of EMR Emissions of EMR Scattering or Refraction of EMR Rotation of EMR UV-Visible IR NMR X-Ray Spectroscopy Fluoroscence Phosphorescence Flame Photometry Nephlometry Turbidometry Raman Spectroscopy Refractrometry Polarimetry 35 Alliance Electro Chemical Methods : Electro Chemical Methods pH metry & Potentiometry Conductometry Voltametry Polarography Amperometery Coulometry 36 Alliance Miscellaneous methods : Miscellaneous methods Thermometric Analysis: DSC Thermo Gravimetric Analysis 37 Alliance Slide 38: Classification of Analytical Methods 1. Classical methods Qualitative – identification by color, indicators, boiling or melting points, odors Quantitative – mass or volume (e.g. gravimetric, volumetric) Based on goal of application: 38 Alliance Slide 39: 2. Instrumental methods Qualitative – chromatography, electrophoresis and identification by measuring physical property (e.g. spectroscopy, electrode potential) Quantitative – measuring property and determining relationship to concentration (e.g. spectrophotometry, mass spectrometry) Often, same instrumental method used for Qualitative and Quantitative analysis. 39 Alliance Slide 40: Types of Instrumental Methods Radiation emission Emission spectroscopy (X-ray, UV, visible), fluorescence, phosphorescence, luminescence Radiation absorption Absorption spectroscopy spectrophotometry, photometry, NMR electron spin resonance 40 Alliance Slide 41: Signal Example Method Radiation Scattering Raman spectroscopy Radiation refraction Refractometry Radiation diffraction X-ray and Electron diffraction method Radiation rotation Polarimetry Electrical potential Potentiometry 41 Alliance Slide 42: Signal Example Method Electrical charge Coulometry Electrical current Voltammetry – amperometry polarography Electrical resistance Conductometry Mass Gravimetry Mass-to-charge ratio Mass spectrometry Rate of reaction Flow injection analysis 42 Alliance Slide 43: Chemical and physical properties employed in instrumental methods: 43 Alliance Slide 44: Instruments for Analysis Example: Spectrophotometry Instrument: spectrophotometer Stimulus: monochromatic light energy Analytical response: photocell Data: electrical current Data processor: current meter Readout: meter scale Encoded information 44 Alliance Slide 45: Encoded information: detector : device that indicates changes in environment transducer : device that converts non-electrical to electrical data Non-electrical domains Electrical domains Physical Current (light intensity, colour) Chemical (pH) Voltage 45 Alliance Advantages of Instrumental Methods : Advantages of Instrumental Methods High Sensitivity Accuracy Small Sample can be used Measurements obtained are reliable & Reproducible Fast Determination Complex sample can be handled easily Process can be made automatic 46 Alliance Limitations of Instrumental methods : Limitations of Instrumental methods High Cost Necessary to use reference substance Skilled Persons are required 47 Alliance Slide 48: Selecting an Analytical Method Based on answers to the following questions: What accuracy and precision are required? How much sample is available? What is the concentration range of the analyte? What components of the sample will cause interference? What are the physical and chemical properties of the sample matrix? How many samples are to be analyzed? 48 Alliance IR Spectroscopy : IR Spectroscopy INFRARED REGION : INFRARED REGION 4000cm-1 to 600 cm-1 IR spectrum: Plot of Trasmittance/Absorbance VS Frequency of radiation SAMPLE IR TRANSMITTED ABSORBED 50 Alliance Molecular vibration : Molecular vibration Atoms joined by covalent bonds undergo continual vibrations relative to each other The energies associated with these vibrations are quantitized. Within a molecule only specific vibrational energy levels are allowed. The energies associated with transitions between vibrational energy levels for most covalent bonds are from 2 to 10 kcal/mol (8.4 to 42kj/mol) 51 Alliance Infrared spectroscopy : Infrared spectroscopy Vibrational IR spectral region covers 2.5 x 10-6 (2.5 µm) to 2.5 x 10-5 (25 µm) Absorption of IR radiation in this region causes bond to change from a lower vibrational energy level to a higher one IR SPECTRUM REPRESENTS CHANGES IN ENERGY BROUGHT BY TRANSITION OF MOLECULES FROM ONE VIBRATIONAL OR ROTATIONAL ENERGY STATE TO ANOTHER. 52 Alliance Slide 53: IR causes a vibration only if there is a change in dipole during vibration Therefore symmetric bonds are inactive. CH3 - CH3 will not observe IR stretch for C-C bond of ethane 53 Alliance Infrared (IR) and Raman Spectroscopies : Infrared (IR) and Raman Spectroscopies Infrared (IR) and Raman spectroscopies have been standard methods of analytical pharmacy and chemistry for a long time. IR and Raman spectra, which are complementary to each other, provide images of vibrations of the atoms of a compound. Therefore, both techniques are also referred to as vibrational spectroscopy. An IR spectrum is obtained by passing infrared radiation through a sample and determining what fraction of the incident radiation is absorbed at particular frequency. On the other hand, a Raman spectrum is obtained by focusing monochromatic radiation on a sample and analyzing the scattered light as a function of frequency. IR spectroscopy is based on the absorption of electromagnetic radiation by a molecular system, Raman spectroscopy relies upon inelastic scattering of electromagnetic radiation by a molecular system. 54 Alliance Applications of IR : Applications of IR Fourier Transformed Infrared (IR) and Raman Spectroscopy are important tools for the solid state characterization of pharmaceutical solids and for the identification of their chemical structures. Generally both methods are applied in combination with other methods for solid state characterization of pharmaceutical solids (e.g. X- ray powder diffraction, DSC, TG). characterization of polymorphism and detection in drug product. Both methods are also suitable for the identification/ detection of the polymorphic form in the tablet. 55 Alliance Applications of FTIR : Applications of FTIR Very high resolution required- Gaseous Mixtures Study of samples having very high absorption Study of samples with weak absorption bands Used in protein structure determination Used in characterizing Adrenocarcinoma Very small sample size: Obtaining Reflection spectra IR emission study 56 Alliance Applications of Near IR : Applications of Near IR NIR: good penetration properties Minimal sample penetration required Thick layers can be analyzed Not much useful for identification Quantitative analysis of compounds containing functional groups made of H bonded to O, C, N Determination of 10, 20, 30 amines 57 Alliance Applications of ATR-FTIR : Applications of ATR-FTIR Surface analysis of biological structures Monitoring reaction processes Evaluation of fermented foods 58 Alliance Pharmaceutical applications of Mid-IR and Raman spectroscopy : Pharmaceutical applications of Mid-IR and Raman spectroscopy Mid-IR and Raman spectroscopy are versatile tools in pharmaceutics and biopharmaceutics, with a wide field of applications ranging from characterization of drug formulations to elucidation of kinetic processes in drug delivery. New developments in applications of these methods for studying drug delivery systems. FTIR-ATR is a well-established standard method used to study drug release in semisolid formulations, drug penetration, and influence of penetration modifiers; it is also capable of in vivo studies. 59 Alliance Slide 60: FTIR-PAS (photo acoustic spectroscopy) has been applied to: Measure drug content in semisolid and solid formulations, Determine drug penetration into artificial and biological membranes. Raman spectroscopy can be used to: Characterize the structure of colloidal drug carrier systems. In vivo studies. Recently, there has been tremendous technical improvement in vibrational microspectroscopy: FTIR imaging shows great promise in its ability to visualize the drug and excipient distribution in pharmaceutical formulations such as tablets and therapeutic transdermal systems, as well as to reveal the mechanism of drug release. Furthermore, this unique technique offers completely new possibilities to study the lateral diffusion of drugs. 60 Alliance Slide 61: NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY -an ideal tool for Diagnosis & Drug Design 61 Alliance INTRODUCTION : INTRODUCTION BASIC PRINCIPLE IN NMR Atomic Nuclei – tiny magnets In external Magnetic Field - allign or oppose If irradiated with EMR of proper frequency – Resonance √ α βo PARAMETERS Chemical shifts Spin lattice relaxation time 62 Alliance Slide 63: NUCLEAR SPIN STATES - HYDROGEN NUCLEUS + 1/2 - 1/2 The two states are equivalent in energy in the absence of a magnetic or an electric field. + + The spin of the positively charged nucleus generates a magnetic moment vector, m. m m TWO SPIN STATES 63 Alliance Slide 64: 64 Alliance Slide 65: RESONANCE Absorption of energy by the spinning nucleus 65 Alliance Resonance : Resonance Resonance: Matching of natural frequency with the applied frequency. In NMR the applied radio frequency has to be match with the precessional frequency of nuclei (which is the consequences of applied external magnetic field) Absorption of radio frequency occur by nuclei after resonance is achieved. 66 Alliance Slide 67: Precessional frequency (n) The number of revolution per second made by magnetic moment vector of the nucleus around the external magnetic field B0 OR Precessional frequency is equal to the frequency of electromagnetic radiation in MHz (Mega cycles/sec) necessary to induce a transition from one spin state to other. 67 Alliance Nuclear Spin Energy Levels : Nuclear Spin Energy Levels Bo +1/2 -1/2 In a strong magnetic field (Bo) the two spin states differ in energy. aligned unaligned N S 68 Alliance Absorption of Energy : Absorption of Energy Bo +1/2 -1/2 +1/2 -1/2 DE = hn DE quantized Radiofrequency Applied Field Aligned Opposed 69 Alliance Slide 70: Bo DE + 1/2 - 1/2 = kBo = hn degenerate at Bo = 0 increasing magnetic field strength THE ENERGY SEPARATION DEPENDS ON Bo 70 Alliance The Larmor Equation : The Larmor Equation g n = 2p Bo g is a constant which is different for each atomic nucleus (H, C, N, etc) DE = kBo = hn can be transformed into gyromagnetic ratio g strength of the magnetic field frequency of the incoming radiation that will cause a transition n α Bo Stronger magnetic fields (Bo) cause the instrument to operate at higher frequencies (n). 71 Alliance NMR Signals : NMR Signals The number of signals shows how many different kinds of protons are present. The location of the signals shows how shielded or deshielded the proton is. The intensity of the signal shows the number of protons of that type. Signal splitting shows the number of protons on adjacent atoms. 72 Alliance DIFFERENT TECHNIQUES : DIFFERENT TECHNIQUES Based on nuclei H1 NMR C13 NMR F13 NMR & P31 NMR N15 NMR & O17 NMR CWNMR (continuous wave NMR) FTNMR 2D_NMR &3D_NMR COSY _CORRELATION SPECTROSCOPY MRI_MAGNETIC RESONANCE IMAGING ESR_ELECTRON SPIN RESONANCE SPECTROSCOPY 73 Alliance APPLICATIONS OF NMR IN MEDICINE : APPLICATIONS OF NMR IN MEDICINE CLINICAL APPLICATION OF PROTON IMAGING IN DIAGNOSIS BRAIN Distinguishing gray matter & white matter Imaging posterior fossae, brain stem, spinal cord Detect demyelinating lesions, tumors, hemorrhages, infarctions Slide 75: ABDOMEN Metabolic liver disease Measures liver iron over load in hemochromatosis Focal areas of inflammation in chronic active hepatisis KIDNEYS Distinguishing renal cortex & medulla To evaluate transplanted kidney PELVIS Differentiates between Benign prostatic hyperplasia & prostatic carcinoma Detects bladder tumours 75 Alliance Slide 76: LUNG Assessing abnormalities Respiratory gating HEART Tomographic images of heart muscle, chambers, valvular structures ECHO-PLANAR TECHNIQUE Discrimination between infarcted, ischemic & normal myocardium BREAST 3D-NMR & single-slice planar imaging in detecting breast abnormalities 76 Alliance : MUSCULO SKELETAL SYSTEM Demonstrates Osteo myelitis, tumor metastasis in vertebral bodies & pelvic bones Images of muscles, tendons, ligaments BLOOD VESSELS & FLOW Atherosclerotic vascular disease Assess blood flow in major vessels. INVIVO SPECTROSCOPY Chemical shift phenomenon Diagnosis of rare disease to inborn errors- Mc Ardle’s syndrome INVIVO Analysis of Bone flouride content. NMR IMAGING STUDIES : NMR IMAGING STUDIES Eliminates risk of x-radiation Excellent spatial & contrast resolution Detecting diseases at earlier stages 78 Alliance NMR IN PHARMACEUTICAL RESEARCH : NMR IN PHARMACEUTICAL RESEARCH Leading technology for 3-D structure determination of bio-macromolecules Studying protein structure SAR by NMR – Novel lead compounds Chemical shift mapping – Structural information on the binding modes and site positions Molecular dynamics, conformational analysis SHAPES – Lead generation NMR – SOLVE 79 Alliance Slide 80: Structural genomics Identifying gene products in disease Targets of drug design. Development of Novel drug delivery systems Enhancing HTS assays Thousands of compounds Cryogenic NMR technology / cryoprobes 80 Alliance Slide 81: 1.Identification of structural isomers 2.Detection of hydrogen bonding 3.Detection of aromaticity 4.Distinction between cis and trans isomer 5.Detection of electronegative atom/group 6.Detection of some double bond character due to resonance 7.Importance in quantitative analysis 81 Alliance NMR Nobel Prize Laureates : NMR Nobel Prize Laureates The Nobel Prize in Physics 1943, Ohostern, USA – Discovery of magnetic moment of proton The Nobel Prize in Physics 1952- IsidorJ. Rafi,USA – Discovery of new methods for nuclear magnetic precision The Nobel Prize in Chemistry 1991 – Felix Bloch, USA and Edward M. Purcell, USA – Methodology of high resolution NMR. The Nobel Prize in Chemistry 2002 – Kurt Wuthrich, Switzerland – Determining 3-D Structure by NMR. The Nobel Prize in Medicine 2003 – Paul C. Lauterbur, USA and Peter Mans field, U.K. – Discoveries concerning MRI. 82 Alliance MASS SPECTROMETRY : MASS SPECTROMETRY 83 Alliance Introduction : Introduction Mass spectrometry is employed to analyze combinatorial libraries, sequence biomolecules, Structure elucidation of unknowns, Environmental and forensic analytics, Quality control of drugs, flavors and polymers 84 Alliance Slide 85: The basic principle of mass spectrometry (MS): To generate ions from either inorganic or organic compounds by any suitable method, To separate these ions by their mass-to-charge ratio (m/z) And to detect them qualitatively and quantitatively by their respective m/z and abundance. Besides electrons, (atomic) ions or photons, energetic neutral atoms and heavy cluster ions can also be used to effect ionization of the analyte. The time-of-flight (TOF) analyzer, ion separation by m/z can be effected in field free regions, too, provided the ions possess a well defined kinetic energy at the entrance of the flight path. The analyte may be ionized thermally, by electric fields or by impacting energetic electrons, ions or photons. 85 Alliance Application of LDI (Laser Desorption/Ionization) : Application of LDI (Laser Desorption/Ionization) LDI of peptides and oligosaccharides LDI is much better suited for the analysis of organic and inorganic salts Molecules with large conjugated π-electron systems Organic dyes as contained in ball point inks Porphyrins UV light-absorbing synthetic polymers LDI presents a useful alternative to MALDI in the low-mass range. In addition, solvent free sample preparation can be employed with insoluble analytes. However, LDI is a “harder” method than MALDI and fragmentation 86 Alliance Applications of MALDI : Applications of MALDI MALDI-MS of Synthetic Polymers: Fingerprints by MALDI-MS Carbohydrates by MALDI-MS Structure Elucidation of Carbohydrates by MALDI Oligonucleotides in MALDI 87 Alliance Slide 88: MALDI is the method of choice for the analysis of synthetic polymers because it usually provides solely intact and singly charged quasimolecular ions over an essentially unlimited mass range. While polar polymers such as poly (methylmethacrylate) (PMMA), polyethylene glycol (PEG), and others readily form [M+H]+ or [M+alkali]+ ions, Nonpolar polymers like polystyrene (PS) non-functionalized polymers like polyethylene (PE) can only be cationized by transition metal ions in their 1+ oxidation state. The formation of evenly spaced oligomer ion series can also be employed to establish an internal mass calibration of a spectrum. The most important parameters that can be determined by MALDI are number-average molecular weight (Mn) weight-average molecular weight (Mw) molecular weight distribution expressed as polydispersity (PD) 88 Alliance Application of Electrospray Ionization : Application of Electrospray Ionization ESI of Small Molecules (polar analytes) Peptide and protein characterization including their complete sequencing ESI is applied to Metal Complexes (ionic) and related compounds ESI of Surfactants: Cationic, anionic and non-ionic surfactants are readily detected by ESI Oligonucleotides, DNA, and RNA are best analyzed by negative-ion ESI ESI of Oligosaccharides and closely related compounds such as glycoproteins, gangliosides, liposaccharides etc. permits molecular weight determination and structure elucidation 89 Alliance LC-MS Applications in drug development : LC-MS Applications in drug development 90 Alliance LC-MS Applications in drug development : LC-MS Applications in drug development 91 Alliance LC-MS Applications in Drug Discovery : LC-MS Applications in Drug Discovery 92 Alliance Peptide Mapping : Peptide Mapping Peptide Mapping: The identification of proteins is essential for understanding biological process and the function of the protein during normal and disease states. The resulting insights lead to the development of therapies for intervention, and ultimately, the cure of disease. The information and knowledge derived from this type of study are extremely valuable for activities involved with target identification activities during drug discovery. Two-dimensional gel electrophoresis (2-DGE) is the primary analysis tool used in the pharmaceutical industry to characterize the expression of proteins. Mass spectrometry-based methods are emerging as an important partner with 2-DGE for correlating proteins to their function. 93 Alliance Peptide Mapping : Identification schemes that involve LC/MS and MALDI-TOF techniques have been applied to a broad range of events that involve proteins such as covalent modification, proteolytic cleavage, and binding. Usually, the focus of LC/MS methods for protein characterization is on four distinct goals: (1) confirmation of putative sequence; (2) identification of amino acid modifications; (3) identification of known proteins; (4) sequence determination of unknown proteins. 94 Peptide Mapping Alliance Glycoprotein Mapping : Glycoprotein Mapping Similar analysis strategies can be applied for the peptide mapping of glycoproteins combined in-source collisionally induced dissociation (CID) with LC/MS/MS to identify sites of N- and O-linked glycosylation. This novel approach uses a series of LC/MS and LC/MS/MS experiments to generate peptide maps and to selectively screen for glycoproteins. The method is based on the characteristic fragmentation of glycoproteins to form a N-acetylhexosamine (HexNAc+) fragment ion at m/z 204. This fragment ion serves as a diagnostic marker for N- and O-linked glycopeptides. 95 Alliance Slide 96: A mixture of peptides and glycopeptides is generated when the glycoprotein is reduced, alkylated, and enzymatically digested. A series of three separate experiments, is used to identify the N- and O-linked glycopeptides. The first experiment uses LC/MS to provide a map of the peptide portion of the protein and to indicate the presence of glycopeptides. The second experiment involves the use of LC/MS/MS to screen the mixture for compounds that fragment to yield the diagnostic m/z 204 (HexNAc+). N- and O-linked glycopeptides are both identified. A final experiment is performed on a sample treated with peptide N:glycosidase F (PNGase F) to release the N-linked oligosaccharides and to identify the O-linked glycopeptides exclusively. 96 Alliance Slide 97: 97 Alliance Slide 98: Additional structural analysis of glycoproteins generally requires fragmentation by chemical or enzymatic cleavage and by separation or isolation. On-line LC/MS methods provide an integrated approach for this type of analysis. 98 Alliance Natural Products Dereplication : Natural Products Dereplication Natural products offer a source of unique chemical diversity for the pharmaceutical industry. Drugs derived from natural products have been introduced for the treatment of cancer, immunosuppression, cardiovascular therapy, and anti-infective therapy. Most antibiotics come from secondary metabolites of soil microorganisms that inhibit bacteria or fungi. Large scale screening of microorganism fermentations followed by isolation and structure elucidation is required. Because many natural products have been previously identified, approaches that avoid time-consuming isolation and provide quick elucidation are essential. 99 Alliance Slide 100: a new strategy was introduced by Ackermann et al. that uses an on-line ESI±LC/MS approach that integrates multi-component identification, fraction collection, and sample preparation for bioactivity screening (1996). Using this LC/MS based approach, crude extracts are screened without extensive purification and chemical analysis. Less material is required due to the sensitivity of the technique and chromatographic resolution is retained. This resolution would ordinarily be relatively poor with a fraction collection process. Furthermore, because the ESI acts as a concentration- dependent detector, the HPLC effluent can be split between mass spectrometry analysis and fraction collection. Thus, molecular weight information is obtained, and approximately 90% of the fractionated material is recovered for biological testing. 100 Alliance Slide 101: 101 Alliance Bioaffinity Screening : Bioaffinity Screening Combinatorial chemistry has changed the strategy of drug candidate synthesis. As a result, hundreds of thousands of compounds are now screened against a particular biological target. Once activity is determined for a mixture, the identification of the active component(s) is necessary. However, this approach requires a considerable amount of resources. To reduce the time and resources required for screening the large number of compounds produced by combinatorial chemistry, approaches featuring the parallel screening of mixtures of compounds (20±30) have been investigated. The recent studies performed by Anderegg and coworkers describe the use of bioaffinity selection LC/MS methods for the identification of active mixture component(s) (Davis et al., in press). This approach features an integrated bioaffinity based LC/MS screening method to separate and identify Lead compounds from mixtures. 102 Alliance Slide 103: 103 Alliance In Vivo Drug Screening : In Vivo Drug Screening Technology for automated highthroughput bioanalysis. focused on methods for determining multicomponent mixtures of drug candidates that involve pharmacokinetics and metabolic stability. These activities were performed during the preclinical and clinical stages of drug development. The need to provide an early assessment of these properties resulted in new strategies for in vivo drug screening during the drug discovery stage. LC/MS/MS approaches, using relatively simple isolation and chromatography conditions, present an effective approach to evaluate new lead compounds for subsequent development or selection of an optimal drug candidate within a specific therapeutic area or a targeted class of compounds. 104 Alliance Metabolic Stability Screening : Metabolic Stability Screening Recently, the use of fast gradient elution LC/MS techniques was described for high throughput metabolic stability screening (Ackermann et al., 1998). The method uses a HPLC column-switching apparatus to desalt and analyze lead candidates incubated with human liver microsomes. The rapid structure identification of metabolites using LC/MS/MS with an ion trap mass spectrometer (Lopez et al., 1998). In this study, metabolic stability analysis is automated to provide maximum structural information in combination with predictive strategies for biotransformation. 105 Alliance Slide 106: This feature is unique and avoids the multiple (2±4) injections that are necessary with other MS/MS configurations (e.g., tandem quadrupole). Along with the significant savings in time, detailed structure information is generated, which enables a comprehensive analysis of substructure relationship to be constructed for each metabolite. These automated studies provide unique advantages during drug discovery, and provide an early perspective on the metabolically labile sites, or ``soft spots'' of a drug candidate. This knowledge is useful during lead optimization activities, and can lead to the initiation of proactive research efforts that deal with metabolism-guided structural modification and toxicity. 106 Alliance Applications of LC/MS in Preclinical Development. : Applications of LC/MS in Preclinical Development. 107 Alliance Application of LC/MS in Clinical Development : Application of LC/MS in Clinical Development 108 Alliance Applications of LC/MS in Manufacturing : Applications of LC/MS in Manufacturing 109 Alliance Applications of LC-MS : Applications of LC-MS Toxicological studies, finding drugs and toxins in variety of materials with smaller sample volume Urine analysis for toxicology Dope test: doping agents and drugs of abuse (steroids, diuretics) Impurity profiling Metabolite studies Bio-equivalence and Bioavailability studies Molecular weight Assay of drugs, intermediates etc. 110 Alliance