Biotransformation And Elimination Of Drugs Biology Essay

Biotransformation And Elimination Of Drugs Biology Essay Liver plays a significant role in the metabolism of a large number of drugs and toxins. Hepatic drug metabolism involves various processes, broadly classified as Phase 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 many endogenous substrates, like bilirubin, steroids etc., by increasing the hydrophilicity and clearance. UGTs are versatile enzymes, in terms of broad, yet overlapping substrate specificity, presence of numerous isoforms, genetic polymorphisms etc. Biological systems are recognised to be stereoselective in nature. A large number of drugs, nearly 50% of all marketed drugs exist as either single enantiomers or racemates. Therefore stereoselective behaviour of drugs plays an important role in drug action as well as disposition (Absorption, Distribution, Metabolism and Elimination). The aim of this project 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. Furthermore, predicting the enantiomeric behaviour of drugs may also aid in rationalising in-sillico modelling of drug metabolism and thereby predicting metabolism of new chemical entities (NCEs) 1. INTRODUCTION Biotransformation and elimination of drugs from the body involves several different metabolic routes. These metabolic pathways are broadly classified 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 reactions like, oxidation, reduction, hydrolysis, isomerisation and so on depending on the chemical nature of the drug. These reactions are catalysed by enzymes such 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 phase 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 prevalent conjugation reaction in the body. [1] 2. GLUCURONIDATION Glucuronidation 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 primarily due to the abundance in living systems of UDP-glucuronic acid, the co-factor for the reaction, as well as due to the pervasive nature of the enzyme, UDP-glucuronosyl transferases (UGTs). [1] [2] The process of glucuronidation involves: I. Formation of co-factor (UDP-glucuronic acid) II. Conjugation of UDP-glucuronic acid with substrate The formation of co-factor (UDP-glucuronic acid) This consists of a two step process: 1. Formation of UDP-glucose Glucose-1-phosphate is present in high concentrations in almost all cells of the body. The first stage of glucuronidation is related 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] [3] 2. Dehydrogenation of UDP-glucose to UDP-glucuronic acid The above step is followed by dehydrogenation of UDP-glucose to UDP-glucuronic acid, catalysed by the enzyme UDP-glucose dehydrogenase, in the presence of NAD+ co-factor. [3] Conjugation of the substrate with UDP-glucuronic acid Conjugation reaction involves transfer of one ÃŽÂ ±-D-glucuronic acid moiety 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 nucleophilic substitution (SN2), whereby the C1 carbon of glucuronic acid, which is in ÃŽÂ ±-configuration, during its reaction with the substrate inverts to form a ÃŽÂ ²-D-glucuronide. [3] [4]. The glucuronide formed is excreted via urine or bile, depending on the chemical nature and molecular weight of the conjugate. [1] [2] The entire reaction is summarised below: Figure: 1: Glucuronidation Pathway leading to formation of ether glucuronide. [3] 2.1 PHARMACOLOGICAL RELEVANCE Based on the functional group of the substrate molecule, the following types of glucuronide conjugates may be formed; [1] O-Glucuronide They 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. Figure: 2 O- Glucuronidation of Morphine [5] [6] UGT Enzyme UDPGA N-glucuronide They are formed by the reaction of UDP-glucuronic acid (UDPGA) with amines, amides etc. E.g. Sulfanilamide, Cyproheptidine, Dapsone S-Glucuronide Reaction of thiol groups with UDPGA in presence of UDP-gucuronosyl transferase results in S-glucuronides. E.g.Disulfiram, 2-Mercapto benzothiazole C-glucuronide It is an uncommon metabolic pathway that occurs due to the direct attachment of UDPGA to the carbon skeleton of drugs. E.g. Sulfinpyrazone [1] [3] 3. UDP-GLUCURONOSYL TRANSFERASE (UGT) ENZYMES 3.1 SITE UGT enzymes are present in human beings 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. Inside 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. [1] 3.2 STRUCTURAL ASPECTS UDP-glucuronosyl transferase enzyme does not contain a prosthetic group. The monomeric molecular weight of the enzyme if found to be between 50- 60 kilo 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 published (by Miley et.al. 2007) [1] [2] 3.3 PHYSIOLOGICAL RELEVANCE In 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] [8] 3.4 MULTIPLE FORMS Various forms of UDP-glucuronosyl transferase (UGT) enzymes have been identified with the help of studies based 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 amino 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 greater than 60%. [1] [2] Nomenclature Divergent evolution and sequence similarity forms the basis of nomenclature of UGT enzymes. Name of the enzyme consists of 4 parts: [1] Root Symbol The root symbol UGT stands for UDP- glucuronosyl transferase. Family It is denoted by Arabic number. E.g. 1, 2 etc Sub-family Designated by an upper-case alphabet Individual Form An Arabic numeral is used for unique identification of the individual form of the enzyme. E.g. UGT2B4, UGT1A6 [1] Mammalian 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 contain 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] [2] UGT2 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. UGT2A2 in liver and small intestine, while UGT2A3 in small intestine, liver and adipose tissue. [2] Figure: 3: Shows the Phylogenetic tree of different UGT isoenzymes. %values indicate the homology between two groups or single isoenzymes at the amino-acid level. [9] 3.5 TISSUE SPECIFICITY The 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. [1] 3.6 SUBSTRATE SPECIFICITY UGTs 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] [6] Table: 1: Showing Substrate Specificity of UGT enzyme isoforms. [6] Sl.No Substrate Max. Specific enzyme activity / pmol.min-1.mg protein-1 1A1 1A3 1A4 1A6 1A8 2A1 2B4 2B7 2B15 2B17 1. Phenols 1900 239 30 2400 5300 735 0.4 5 167 38 2. Amines 1 84 540 10600 1800 22 3 0 3 Opioids 0 130 0 0 0 73 0 3462 0 4. Carboxylic acids 0 121 0 170 68 0 2 0 5. Bilirubin 400 0 2 0 0 0 0 0 0 3.7 INTERINDIVIDUAL VARIATIONS Several 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. [1] 4. ENZYME KINETICS: Study of enzyme kinetics helps to understand the catalytic mechanism of the enzyme; role played by the enzyme in metabolism as well the rate and activity of enzyme. Michaelis Menten equation is used to describe enzyme substrate interaction and is given by: [2] k1 k 2 E + S ES E + P k-1 Where E = Enzyme, S = Substrate, P = Product Michaelis Constant Km is given by: Km = (k 2 + k -1) / k 1 Michaelis 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 typical model of Michaelis Menten plot is shown below: [2] Figure: 3 Michaelis Menten hyperbolic kinetic profile. [10] Here Vmax is the maximum velocity of enzyme action. Vmax / Km is an indicator of the catalytic efficiency of the enzyme. 5. STEREOISOMERISM Molecules 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. Stereoisomers 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] [11] While 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. [2] 5.1 DRUGS AS ENANTIOMERS As discussed above, the pharmacokinetic and pharmacodynamic properties may vary for each individual enantiomer. In 1992, United States Food and Drug Administration (US FDA) published 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] [12] Some examples of some single enantiomeric drugs which have gained importance, compared to their racemate counterparts are given below, due to their improved pharmacodynamic- pharmacokinetic profiles: L-DOPA: The use of levo dopa instead of racemic dopa has resulted in reduction in dose and adverse effects (nausea, vomiting, anorexia, granulocytopenia) Figure: 4: Levodopa [13] ESOMEPRAZOLE: This proton-pump inhibitor, which is the S-enantiomer of Omeprazle has shown lower first pass effect and higher 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 [14] LEVOFLOXACIN: It 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 [15] R-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 effects Figure: 7: R-Salbutamol [16] R, R-METHYLPHENIDATE: This 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 rapid onset of action and reduced adverse effects Figure: 8 R, R-Methylphenidate [17] 6. AIM OF THE PROJECT This 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 liquid chromatography (HPLC) equipped with a mass spectrometer for detection. [14] Laboratory analysis of enantiomers is usually done using any one of the following two methods: Chiral Chromatography, which make use of a chiral column or chiral mobile phase 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 DIRECTIONS Many 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 Structure Metabolism Relationship (2D- QSMR), 3D-Quantitative Structure Metabolism Relationship (3D- QSMR), Pharmacophore Identification as well as Non-linear pattern recognition 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 widely accepted, development of effective models for UDP-glucuronosyl transferases (UGTs) catalyzed Phase 2 metabolism has received much less attention. [15] Versatility 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, apart from a few catalytically relevant amino acids, the full X-ray crystal structure of UGT enzyme is not yet elucidated. [15] [18] Depending 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, study of electronic nature of the nucleophile and pKa is also significant. Since chirality plays an important role in determining metabolic behaviour of drugs, design 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] [17] 8. CONCLUSION In 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, knowing the enantiomeric behaviour may help in the development of 3D-Quantitative Structure Metabolism Relationship (3D-QSMR) models for predicting drug metabolism.