Biotransformation And Elimination Of Drugs Biology Essay

Biotransformation And excreta Of Drugs Biology EssayLiver plays a significant role in the transfiguration of a astronomical tot up of do do medicinesss and toxins. Hepatic drug metamorphosis involves assorted processes, broadly sort as flesh 1 (functionalisation) and Phase 2 (conjugation). Glucuronidation catalysed by UDP-glucuronosyl transferase (UGTs) plays a key role in the Phase 2 metabolism of a large number of drugs as well as m all a nonher(prenominal) endogenic substratums, homogeneous bilirubin, steroids etc., by increasing the hydrophilicity and clearance. UGTs be versatile enzymes, in terms of broad, yet overlapping substrate specificity, mien of many isoforms, genetic polymorphisms etc.Biological systems atomic number 18 recognised to be stereoselective in nature. A large number of drugs, nearly 50% of all marketed drugs exist as either single enantiomers or racemates. thence stereoselective deportment of drugs plays an important role in drug exercise as well as disposition (Absorption, Distribution, metabolous process and Elimination).The aim of this throw off is to identify the enantio-selectivity of drugs towards glucuronidation by UGT and also to identify whether enantio-selectivity is linked to specific isoforms of UGT. Further more than, predicting the enantiomeric behaviour of drugs may also aid in rationalising in-sillico modelling of drug metabolism and thereby predicting metabolism of impertinent chemic entities (NCEs)1. INTRODUCTIONBiotransformation and elimination of drugs from the organic building involves several dissentent metabolic routes. These metabolic pathways atomic number 18 broadly classified advertisement into Phase 1 (functionalisation) and Phase 2 (conjugation) drug metabolism.Phase 1 metabolic pathway involves transforming the drug into a more polar functionality, through various answers like, oxidation, reduction, hydrolysis, i whateverrisation and so on depending on the chemical substance nat ure of the drug. These reactions are catalysed by enzymes such(prenominal) as Cytochrome P450, NADPH-cytochrome P450 reductase, acetlycholineestrase etc.Product of Phase 1 drug metabolism may then act as a substrate for Phase 2 metabolism. This mannikin consists of conjugation of the drug substrate with endogenous ligands leading to increased polarity, hydrophilicity and thereby elimination of the drug from body through bile or urine. Conjugation reactions include glucuronidation, glycosidation, sulfation, methylation etc. These reactions are catalysed by UDP-Glucuronosyltransferase, UDP-Glycosyltransferase, Sulfotransferase, Methyltransferase respectively. Among these, glucuronidation is the most universal conjugation reaction in the body. 12. GLUCURONIDATIONGlucuronidation is the most common reaction in Phase 2 drug metabolism. This conjugation reaction which is catalysed by UDP- glucuronosyl transferase, forms about 35% of all drugs metabolised by conjugation. This is primaril y repayable to the teemingness in living systems of UDP-glucuronic window pane, the co-factor for the reaction, as well as due to the pervasive nature of the enzyme, UDP-glucuronosyl transferases (UGTs). 1 2The process of glucuronidation involvesI. organic law of co-factor (UDP-glucuronic acid)II. Conjugation of UDP-glucuronic acid with substrateThe formation of co-factor (UDP-glucuronic acid)This consists of a two step process1. Formation of UDP-glucoseGlucose-1-phosphate is dumbfound in high concentrations in almost all cells of the body. The premier(prenominal) stage of glucuronidation is re juveniled to glycogen synthesis through the common intermediate, UDP-glucose. The formation of UDP-glucose occurs by addition of a Uridine 5- diphosphate (UDP), a pyrophosphate nucleotide in cells, to a molecule of Glucose-1-phosphate. The reaction is catalysed by UDP-glucose pyrophosphorylase enzyme. 1 32. Deenthalpyation of UDP-glucose to UDP-glucuronic acidThe above step is followe d by dehydrogenation of UDP-glucose to UDP-glucuronic acid, catalysed by the enzyme UDP-glucose dehydrogenase, in the presence of NAD+ co-factor. 3Conjugation of the substrate with UDP-glucuronic acidConjugation reaction involves transfer of one -D-glucuronic acid mediety from the co-substrate UDP-glucuronic acid (UDPGA), which act as an energy rich intermediate, to form the glucuronide conjugate of the drug molecule. The reaction is catalysed by UDP-glucuronosyl transferase (UGT) enzyme.The reaction is found to be a bimolecular(a) nucleophilic substitution (SN2), whereby the C1 carbon of glucuronic acid, which is in -configuration, during its reaction with the substrate inverts to form a -D-glucuronide. 3 4. The glucuronide form is excreted via urine or bile, depending on the chemical nature and molecular free weight of the conjugate. 1 2The entire reaction is summarised below flesh 1 Glucuronidation Pathway leading to formation of ether glucuronide. 32.1 pharmacologic RELEVAN CEBased on the functional group of the substrate molecule, the following types of glucuronide conjugates may be formed 1O-GlucuronideThey are formed from Phenols, alcohols, carboxylic acids. O-gulcuronides are chiefly excreted in to bile and may undergo entero-hepatic circulation. Examples of drugs Morphine, Chloramphenicol, Salicylic acid, Clofibrate. visualise 2 O- Glucuronidation of Morphine 5 6UGT EnzymeUDPGAN-glucuronideThey are formed by the reaction of UDP-glucuronic acid (UDPGA) with amines, amides etc. E.g. Sulfanilamide, Cyproheptidine, DapsoneS-GlucuronideReaction of thiol groups with UDPGA in presence of UDP-gucuronosyl transferase results in S-glucuronides. E.g.Disulfiram, 2-Mercapto benzothiazoleC-glucuronideIt is an uncommon metabolic pathway that occurs due to the direct attachment of UDPGA to the carbon brief of drugs. E.g. Sulfinpyrazone 1 33. UDP-GLUCURONOSYL TRANSFERASE (UGT) ENZYMES3.1 SITEUGT enzymes are present in human macrocosms and most other mammals. The enzyme is located in many tissues of the body, mostly in liver but also in kidney, lungs, small intestine, spleen, adrenals and skin, to a lesser extend. in pique of appearance the cell, UGTs are bound to the membranes of endoplasmic reticulum. Most of the Phase 1 metabolic enzymes, including cytochrome P450s, are located in the endoplasmic reticulum. Therefore endoplasmic reticulum is regarded as an ideal site for UGT enzymes, as it facilitates glucuronide conjugation of Phase 1 substrates. 13.2 STRUCTURAL ASPECTSUDP-glucuronosyl transferase enzyme does not acquit a prosthetic group. The monomeric molecular weight of the enzyme if found to be between 50- 60 kilogram Daltons. The protein sequence of the enzyme shows slight variations between each individual form.A full length crystal structure of UGTs is yet to be resolved, although crystal structure of the binding domain for UDP-glucuronic acid in human UGT2B7 has been publish (by Miley et.al. 2007) 1 23.3 PHYSIOLOGICAL R ELEVANCEIn addition to being a major enzyme involved in Phase 2 drug metabolism, UGT enzymes play a number of other roles in the body. Many endogenous compounds such as bilirubin, steroid hormones (e.g. thyroxine, triiodothyronine) and catechols (derived from catecholamine metabolism), act as substrates for UGT enzymes. All these compounds are potentially hazardous if accumulated in the body.Deficiency of UGT enzyme results in hyperbilirubinaemia. Hereditary diseases like Gilberts syndrome and Cringler-Najjars syndrome are associated with genetic polymorphisms of UGT gene 7. Apart from disposition of endogenous toxins, the enzyme also catalyses glucuronidation of various exogenous chemicals and helps in bodys defence against toxic principles 1 83.4 MULTIPLE FORMSVarious forms of UDP-glucuronosyl transferase (UGT) enzymes name been set with the help of studies base on purification, characterization of enzymes, molecular cloning, DNA sequencing etc. About 50 vertebrate UGTs have been identified among which 19 are found in humans.UGT enzymes are divided in to families and sub-families based on similarity of their aminic acid sequences. Two enzymes are in the same family if the similarity of their amino acid sequences is more than 50% and will be grouped into the same subfamily is similarity is niftyer than 60%. 1 2NomenclatureDivergent evolution and sequence similarity forms the basis of nomenclature of UGT enzymes. Name of the enzyme consists of 4 split 1Root SymbolThe root symbol UGT stands for UDP- glucuronosyl transferase.FamilyIt is denoted by Arabic number. E.g. 1, 2 etcSub-familyDesignated by an upper-case alphabet exclusive FormAn Arabic numeral is used for unique identification of the individual form of the enzyme.E.g. UGT2B4, UGT1A6 1Mammalian UGTs are divided in to four families UGT1, UGT2, UGT3 and UGT8. Among these, only UGT1 and UGT2 catalyses conjugation of glucuronide and hence are discussed further.UGT1A family of enzymes are found to co ntain 9 functional proteins and are coded for by a single gene complex located at chromosome 2q37. The genes coding for this enzyme have common exons 2-5 (region of gene which codes for the carboxyl terminus of the enzyme) and a variable exon 1. The first exon is responsible for coding the N-terminal domain of the protein and this explains why the enzymes are substrate specific in spite of have a common C-terminal 1 2UGT2 enzyme family, especially UGT2B plays a vital role in the metabolism of xenobiotics and endogenous ligands. Genes coding for UGT2 family enzymes are situated on chromosome 4q13. In the case of UGT2B sub family, protein sequences at the C-terminal, gives rise to the UDP-glucuronic acid binding domain as well as helps in anchoring of the protein to membrane of endoplasmic reticulum.UGT2A subfamily is less studied and do not play a significant role in systemic metabolism. UGT2A1 is present in olfactory epithelia and to a lesser extend in cells of brain and lungs. UGT 2A2 in liver and small intestine, while UGT2A3 in small intestine, liver and adipose tissue. 2Figure 3 Shows the Phylogenetic tree of different UGT isoenzymes. %values render the homology between two groups or single isoenzymes at the amino-acid level. 93.5 TISSUE SPECIFICITYThe various forms of UGT enzymes show tissue specificity in man. Majority of these enzymes occur predominantly in the liver, (E.g. UGT 1A1, 1A4, 1A6, 2B7 etc) while some others are found in various extrahepatic sites. An example is UGT1A10, which is present in the cells of all areas of gastrointestinal tract and hence accounts for its wide range of substrate specificity, from phenol molecules to steroids. 13.6 SUBSTRATE SPECIFICITYUGTs show a wide, yet overlapping, range of specificity towards drugs and endogenous ligands. For example, glucuronidation of bilirubin is preferred by UGT1A1 and that of morphine by UGT2B7. 1 6Table 1 Showing Substrate Specificity of UGT enzyme isoforms. 6Sl.NoSubstrateMax. Specific enzyme activity / pmol.min-1.mg protein-11A11A31A41A61A82A12B42B72B152B171.Phenols190023930240053007350.45167382.Amines18454010600180022303Opioids0 one hundred thirty000730346204.Carboxylic acids01210one hundred seventy680205.Bilirubin400020000003.7 INTERINDIVIDUAL VARIATIONSSeveral genetic polymorphisms in UGTs may lead to variations between individuals in the ability to glucuronidate drugs and endogenous substrates. Mutations in genes coding for UGT1 enzyme family has been identified as the cause for hereditary hyperbilirubinaemia, characterized by jaundice due to high levels of unconjugated bilirubin in the body.Further, several genetic diseases- Gilberts syndrome and Cringler-Najjars syndrome, may occur due to mutations in genes coding for UGT1A1 isoform. 14. ENZYME KINETICSStudy of enzyme kinetics helps to understand the catalytic mechanism of the enzyme role compete by the enzyme in metabolism as well the rate and activity of enzyme. Michaelis Menten equation is used to dec ipher enzyme substrate interaction and is given by 2k1 k 2E + S ES E + Pk-1Where E = Enzyme, S = Substrate, P = ProductMichaelis Constant Km is given byKm = (k 2 + k -1) / k 1Michaelis Constant Km is an indicator of affinity of substrate for the enzyme as well as the rate of enzyme activity. The kinetics of drug metabolism can also be defined using Michaelis Menten equation and may be plotted in a graph of Rate of reaction (Velocity) vs. Concentration of Substrate. Although not all enzyme substrate reactions are best described by this equation, a characteristic model of Michaelis Menten plot is shown below 2Figure 3 Michaelis Menten hyperbolic kinetic profile. 10Here Vmax is the maximum speed of enzyme action. Vmax / Km is an indicator of the catalytic efficiency of the enzyme.5. STEREOISOMERISMMolecules having the same constitution of atoms and sequence of covalent bond, but differ in their three-dimensional arrangement of atoms in space are known as stereoisomers. Stereoisome rs are classified in to geometrical (cis/trans) isomers, enantiomers and diastereoisomers. Stereoisomers that are mirror images of each other and hence are not superimpossible are called enantiomers. They differ from each other only by one chiral centre. Isomers that are not mirror images are diastereoisomers. They may contain more than one chiral centre. 2 11While geometrical and diastereoisomers are chemically different molecules, enantiomers have identical chemical and physical properties, except for the way in which they rotate plane polarized light. Enantiomers are of great significance in therapeutics as all biological systems represent a chiral environment. Hence drug action as well as disposition (absorption, distribution, metabolism and elimination) may differ between enantiomers. 25.1 DRUGS AS ENANTIOMERSAs discussed above, the pharmacokinetic and pharmacodynamic properties may vary for each individual enantiomer. In 1992, United States Food and Drug Administration (US FD A) produce a policy for development of new stereoisomeric drugs. Approximately 50% of all marketed drugs are found to be racemates. Although many drugs can be safely administered as racemates, some others show better efficacy and fewer side effects when administered as a single enantiomer. For example, cardiac toxicity of the local anaesthetic agent, Levobupivacaine is chiefly associated with R-enantiomer.Further, some drugs undergo chiral inversion inside the body to the other enantiomer (e.g. Ibuprofen Non-steroidal anti-inflammatory agent) and some others undergo racemisation after administration. This is of particular concern, especially if one of the enantiomers is toxic. Hence evaluating drugs for their stereochemistry is gaining importance. 2 12Some examples of some single enantiomeric drugs which have gained importance, compared to their racemate counterparts are given below, due to their improved pharmacodynamic- pharmacokinetic profilesL-DOPAThe use of levo dopa instead of racemic dopa has resulted in reduction in dose and adverse effects (nausea, vomiting, anorexia, granulocytopenia)Figure 4 Levodopa 13ESOMEPRAZOLEThis proton-pump inhibitor, which is the S-enantiomer of Omeprazle has shown lower first suck effect and high plasma half life compared to the R-enantiomer, thus maintaining the intra-gastric pH above 4 for a longer duration. S-enantiomer also showed reduction in variability of response between patients.Figure 5 Esomeprazole 14LEVOFLOXACINIt is a Quinolone antibiotic. As there are slight differences in disposition between enantiomers of this drug, a single S-enantiomer is preferred.Figure 6 Levofloxacin 15R-SALBUTAMOL (LEVALBUTEROL)S-enantiomer has shown increased hyper responsiveness of airway, sensitivity to allergens and some decrease in bronchodilator potency. While R-Salbutamol gives significantly higher bronchodilator effect and lesser side effectsFigure 7 R-Salbutamol 16R, R-METHYLPHENIDATEThis drug is found to be ten-fold more potent than its S-enantiomer when used to treat attention deficit hyperactivity. The presystemic metabolism and disposition of the drug is enantioselective in nature. Further, the R-enantiomer shows quick onset of action and reduced adverse effectsFigure 8 R, R-Methylphenidate 176. AIM OF THE PROJECTThis project aims to determine the rates of glucuronidation of enantiomeric pairs, of a wide range of drugs, to identify differences in metabolism between enantiomers of a drug and also to find out whether enantioselectivity is related to a particular family of UDP-glucuronosyl transferase (UGT) enzyme. Experiment may be done by in-vitro incubations of human recombinant UGTs or human liver microsomes with the selected substrates, followed by analysis using politic chromatography (HPLC) equipped with a mass spectrometer for detection. 14Laboratory analysis of enantiomers is usually done using any one of the following two methodsChiral Chromatography, which make use of a chiral column o r chiral mobile pattern to separate the enantiomers.Derivatisation, of the analyte using a chiral derivative followed by separation of the resulting diastereoisomers using standard, achiral chromatographic method.But in the case of separation of drug conjugates, the analytical process is relatively simple, as the glucuronide conjugates behave just like derivatised diastereomers and hence may be separated by conventional liquid chromatography.7. FUTURE DIRECTIONSMany late stage failures in drug development process are due to inability to predict the pharmacokinetic properties of new chemical entities (NCE) before obtaining data from clinical trials. Hence in-vitro approaches like computational (in-sillico) modelling of drug metabolism is gaining acceptance in the recent times. Many approaches such as 2D-Quantitative twist Metabolism Relationship (2D- QSMR), 3D-Quantitative Structure Metabolism Relationship (3D- QSMR), Pharmacophore Identification as well as Non-linear pattern recog nition techniques are being studied to model drug metabolising enzymes. Although predictive models for metabolism of drugs by the Phase 1 metabolising enzyme, Cytochrome P450 are astray accepted, development of effective models for UDP-glucuronosyl transferases (UGTs) catalyzed Phase 2 metabolism has received much less attention. 15Versatility of these group of metabolic enzymes, in terms of broad but overlapping substrate specificity, drug-drug interactions, genetic polymorphisms as well as presence of a large number of isoforms are some of the challenges facing the development of predictable models for UGTs. Furthermore, asunder from a few catalytically relevant amino acids, the full X-ray crystal structure of UGT enzyme is not yet elucidated. 15 18Depending on the parameters being modelled (e.g. Km, Vmax etc.) a number of physico-chemical and molecular descriptors, such as molecular size, shape, lipophilicity, hydrogen bonding etc., are required to model molecular recognition of substrates and catalysis by UGTs. Apart from this, ascertain of electronic nature of the nucleophile and pKa is also significant. Since chirality plays an important role in determining metabolic behaviour of drugs, soma tools may be developed that address the issue of chirality. While 2D-descriptors will only predict molecular connectivity, 3-D models predicting the enantiomeric properties of enzyme-substrate interactions might significantly improve the future of drug development process. 16 178. CONCLUSIONIn conclusion, many biological systems represent a chiral environment. Therefore assessing the enantioselectivity of drug metabolising enzymes plays a significant role in predicting pharmacokinetic behaviour of drugs. The present project aims at identifying the enantio-selectivity of drugs in UDP-glucuronosyl transferase (UGT) metabolism, which is an important Phase 2 (conjugation) process of drug metabolism. Furthermore, knowledgeable the enantiomeric behaviour may help in the development of 3D-Quantitative Structure Metabolism Relationship (3D-QSMR) models for predicting drug metabolism.