Drug Metabolism & Pharmacokinetics in Drug Discovery: A Primer for Bioanalytical Chemists, Part I Chandrani Gunaratna In the face of advancing technology in combinatorial synthesis and high Bioanalytical Systems Inc. 2701 Kent Avenue throughput screening, the drug discovery process continues to evolve. West Lafayette, IN Preclinical drug metabolism and pharmacokinetics studies play a key role 47906-1382 in lead identification and optimization. This fast-paced development [email protected] process has imposed an enormous burden on the analytical chemist to design faster and more sensitive assay techniques to aid the drug discovery and development. This article, Part I of a two-part series introduces the analytical chemist to the fundamentals of drug metabolism. Part II of this series will discuss the pharmacokinetics aspects and how drug metabolism data can be used to predict pharmacokinetic parameters. Technological innovation and the ciency in the optimization of desired liphophilicity and stability are deter- pressures of competition have pharmacological activity in humans mined by measuring the octanol- caused enormous changes in the while decreasing the reliance on ani- water partition coefficient and pKa. drug discovery process. Progress in mal studies has become a challenge. These measurements are useful in molecular biology and the Human New chemical entities (NCEs) enter predicting the protein binding, tissue Genome Project has contributed to the drug discovery pipeline through distribution and absorption in the the remarkable advances made in combinatorial synthesis and rational gastrointestinal tract (1). identification of new therapeutic tar- drug design where information The selected leads are further gets. The drug discovery process is about the target of action is used to screened using in vitro tests during rapidly evolving due to the techno- design the lead compound. HTS lead optimization. The goal of lead logical developments in target iden- helps the identification of the leads optimization is to select compounds tification along with automation of that provide the required effect at with required biological activity in combinatorial synthesis and high high concentrations. In the secon- humans. Relevant pharmacokinetic throughput screening (HTS). In light dary screening stage physicochemi- parameters such as tissue penetra- of these advances, improving effi- cal properties such as solubility, tion, stability, intestinal absorption, F1 metabolism, and elimination are ob- CHEMICAL COMBINATORIAL Drug development tained using in vitro systems. These process (IND: in vitro systems include mi- Investigational New Lead Identification Drug, NDA: New Drug DISCOVERY crosomes, hepatocytes or tissue Application) slices for metabolite identification HIGH THROUGHPUT SCREENING Lead Optimiz and evaluation of metabolic path- PHYSIOCHEMICAL PROPERTIES inical ways and rates, and caco-2 cell lines l -c IN VITRO SCREENS re for evaluating transcellular absorp- P udies ation t IN VIVO SCREENS tion. Cytotoxicity data can be ob- tion S File IND c tained by using organ-specific cell PHASE I lines. Knowledge of the toxic poten- PHASE II nical ug Intera li r C D tial of these early leads and their PHASE III File NDA possible metabolites is essential for successful drug discovery. Most REGISTRATION drug candidates fail at this stage and only a few will be judged sufficiently MARKET safe and efficacious to proceed fur- ther into development. Both in vitro 17 Current Separations 19:1 (2000) F2 Oral Intestines drug as schematically shown in F3 is Absorption of a drug after known as ADME studies. administration. Topical Skin Drug Metabolism DRUG IV BLOOD Interest in drug or xenobiotic (for- IM, SC, IP Membranes eign compounds) metabolism can be dated back to the early 19th century. Inhalation Lung Metabolism then was known as a “detoxication” mechanism in the F3 body. In late 1930s, with the discov- ery of the synthetic azo-dye Pron- Schematic representation sm Metabolites of drug’s path from blood. Drug at metaboli tosil’s metabolism to antibacterial absorption Drug in Absorption agent sulfanilamide in the body, Site Blood elimination studying metabolism has become an Urine important priority. This year BAS distribution and the International Society for the Other Other Study of Xenobiotics (ISSX) pro- Distribution Tissues Excretory Fluids Fluids duced a historical calendar celebrat- ing many of the original and in vivo studies are then carried drug metabolism and pharmacoki- contributions to our knowledge of out on the active candidate com- netics aspects in drug discovery. Part the metabolism of organic com- pounds. The objective in these pre- 1 of the article covers the basics of pounds (3). clinical studies is not only to identify drug metabolism. In Part II we will Metabolism is the mechanism of the most active leads with the most discuss the kinetics of drug metabo- elimination of foreign and undesir- appropriate safety profiles but also, lism and the relationship of kinetic able compounds from the body and to select the closest animal species to data to the pharmacokinetics of a the control of levels of desirable the human for toxicity studies (2). drug. compounds such as vitamins in the Understanding of pharmacokinetic body. Since information on the me- and metabolism characteristics of Path of a drug tabolism of a drug plays a significant the selected compounds is needed in role in selection and further charac- designing appropriate human clini- After administration by any route, a terization of the drug, an in-depth cal trials. Various stages of drug dis- drug will reach the blood stream as look at the mechanism of drug meta- covery are illustrated in F1. schematically shown in F2.This bolism is worth the effort. Meeting the objectives of drug process is known as absorption. The The major site of metabolism in metabolism research, whether it be drug in the blood distributes rapidly the body is the liver. Metabolism in in vitro or in vivo, requires the proc- between the plasma and blood cells liver occurs in two stages: Phase I essing of a very large number of and also between plasma proteins. pathways in liver microsomes where samples for the determination of Most drugs readily cross the capillar- the drug is functionalized and Phase drug candidates and metabolites. ies and reach the extracellular fluid II pathways in liver cells where the There is likewise a lot of structural of every organ. Lipid soluble drugs parent or the metabolite from Phase chemistry to be done to identify me- cross the cell membranes and distrib- I gets conjugated. Liver microsomes tabolites. It is important for analyti- ute into the intracellular fluid of vari- are in the endoplasmic reticulum of cal chemists to understand that the ous tissues. This process of liver cells or hepatocytes. Phase I vast majority of compounds (actu- transferring a drug from blood to reactions in microsomes are cata- ally > 99.99%) will never become various tissues is called distribution. lyzed by a group of enzymes known drugs. Thus the bioanalytical work A drug is eliminated either di- as the cytochrome P450 system that must be fast. Often elegance must be rectly through an excretory route plays a significant role in drug meta- traded for speed early in the process. such as urine, bile etc. which is bolism. The common chemical reac- Later on, for example in clinical tri- known as elimination; or indirectly tions involved in Phase I are aromatic als, the number of samples for a par- through enzymatic or biochemical hydroxylation, aliphatic hydroxyla- ticular compound will increase transformation by the liver. The lat- tion, oxidative N-dealkylation, oxi- exponentially and carefully vali- ter path of elimination is called me- dative O-dealkylation, S-oxidation, dated methods are both required and tabolism. The study of this whole reduction and hydrolysis. Most often justified. process of absorption, distribution, this simple functionalization could This article is intended to en- metabolism and elimination of a be sufficient to make a drug more lighten bioanalytical chemists on soluble, facilitating elimination www.currentseparations.com 18 T1 toxic and carcinogenic action of Enzyme Substrates Known Cytochrome xenobiotics. P450 substrates. CYP1A2 Amitriptyline, Betaxolol, Caffeine, Clomipramine, Clozapine, There are about 30 human cyto- Chlorpromazine, Fluvoxamine, Haloperidol, Imipramine, chrome P450 enzymes, out of which Olanzapine, Ondansetron, Propranolol, Tacrine, Theophylline, only six, CYP1A2, CYP2C9, Verapamil, (R)-Warfarin CYP2C19, CYP2D6, CYP2E1 and CYP2A6 Coumarin, Betadiene, Nicotine CYP3A4 are the major metabolizing enzymes. CYP3A is the most abun- CYP2C9 Amitriptyline, Diclofenac, Demadex, Fluoxetine, Ibuprofen, Losartan, Naproxen, Phenytoin, Piroxicam, Tolbutamide, dant and most clinically important (S)-Warfarin isozyme in humans. It metabolizes nearly 50% of the clinically available CYP2C19 Amitriptyline, Citalopram, Clomipramine, Diazepam, Imipramine, Omeprazole drugs. T1 shows the major CYPs involved in the metabolism of some CYP2D6 Amitriptyline, Betaxolol, Clomipramine, Codeine, Clozapine, known drugs. From the table it can Desipramine, Fluoxetine, Haloperidol, Imipramine, Methadone, Metoclopramide, Metoprolol, Nortriptyline, Olanzapine, be seen that some drugs are metabo- Ondansetron, Paroxetine, Propranolol, Risperidone, Sertraline, lized by more than one isozyme. This Timolol, Venlafaxine multiple-substrate metabolism is the CYP2E1 Acetaminophen,