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Drugs metabolism and disposition

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  • 1. Drug Metabolism & Pharmacokineticsin Drug Discovery: A Primer forBioanalytical Chemists, Part IChandrani Gunaratna In the face of advancing technology in combinatorial synthesis and highBioanalytical 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 role47906-1382 in lead identification and optimization. This fast-paced developmentprema@bioanalytical.com 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 tained using in vitro systems. TheseDrug developmentprocess (IND: in vitro systems include mi-Investigational New DISCOVERY crosomes, hepatocytes or tissue LeDrug, NDA: New Drug adApplication) slices for metabolite identification Ide HIGH THROUGHPUT SCREENING nti and evaluation of metabolic path- fic Le al PHYSIOCHEMICAL PROPERTIES ati ways and rates, and caco-2 cell lines in ic ad on -c l Op IN VITRO SCREENS for evaluating transcellular absorp- ies Pre tim tu d iza IN VIVO SCREENS tion. Cytotoxicity data can be ob- nS tio n ctio File IND PHASE I tained by using organ-specific cell era lines. Knowledge of the toxic poten- Int al nic PHASE II ug tial of these early leads and their Dr Cli File NDA PHASE III 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 vitro17 Current Separations 19:1 (2000)
  • 2. F2 drug as schematically shown in F3 is Oral IntestinesAbsorption 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 theF3 body. In late 1930s, with the discov-Schematic representation m Metabolites ery of the synthetic azo-dye Pron- bolisof drug’s path from blood. Drug at meta tosil’s metabolism to antibacterial absorption Drug in Absorption agent sulfanilamide in the body, Site Blood eli m studying metabolism has become an ina Urine ti on 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- in g many o f the o riginal 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 o us 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 eliminationwww.currentseparations.com 18
  • 3. T1 toxic and carcinogenic action of Enzyme SubstratesKnown 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, o nly 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, Caffeine, Chlorzoxazone, Dextromethorphan, cause for metabolism-based drug- Ethanol, Theophylline, Venlafaxine drug interactions (DDIs). Some drugs can be inducers or CYP3A4/5 Alprazolam, Amiodaron, Amitriptyline, Astemizole, Bupropion, Buspirone, Caffeine, Carbamazepine, Cerivastatin, Cisapride, inhibitors of specific isozymes but Clarithromycin, Clomipramine, Codeine, Cyclosporine, not necessarily substrates. Enzyme Dexamethasone, Dextromethorphan, DHEA, Diazepam, inducers increase specific enzyme Diltiazem, Donepezil, Doxycycline, Erythromycin, Estradiol, Felodipine, Fluoxetine, Imipramine, Lansoprazole, Lidocaine, levels by modulating the gene ex- Loratadine, Lovastatin, Midazolam, Nicardipine, Nifedipine, pression. Some drugs induce P450 Omeprazole, Orphenadrine, Paroxetine, Progesterone, enzymes that are not involved in their Quinidine, Rifampin, Sertraline, Sibutramine, Sildenafil, Simvastatin, Tacrolimus, Tamoxifen, Terfenadine, metabolism. For example, omepra- Testosterone, Theophylline, Verapami, Vinblastine, (R)-Warfarin zole induces human CYP1A2 but is metabolized by CYP2C19 and through the kidneys. Further conju- Genetic polymorphism CYP3A4 (6). Administration of gation in Phase II occurs by glu- omeprazole can lower the effect of a curonidation, sulfation, amino acid Cytochrome P450 enzymes are drug normally metabolized by conjugation, acetylation, methyla- grouped into families and sub fami- CYP1A2, e.g., acetaminophen. tion or glutathione conjugation to lies based on their structural similar- Enzyme inhibitors function in facilitate elimination. ity (5). Families include CYPs with different ways. The competitive in- >40% amino acid sequence homol- hibitors compete with the substrate Cytochrome P 450 system ogy and are designated by a number for the active site, e.g., fluvoxamine after CYP. Subfamilies are the CYPs and caffeine for CYP1A2 (7). The Cytochrome P450 (CYP) enzyme within a family that have >60% non-competitive inhibitors bind to system, a very large group of en- amino acid sequence homology and the enzyme-substrate complex or to zymes encoded by the P450 gene are designated by a letter following the heme group, e.g., ketoconazole. superfamily, is one of the widely the number. For example CYP3A4 is The third type, irreversible inhibi- studied topics in drug development. a cytochrome P450 enzyme, belong- tors inactivate the enzyme either by CYPs are membrane bound proteins ing to family 3 and subfamily A. The heme binding or protein binding. En- with an approximate molecular last number 4, refers to the sequence zyme inhibition can lead to higher weight of 50 kD, and contain a heme of discovery. systemic levels of a drug causing moiety. CYPs and other mixed func- Several of the drug metabolizing enhanced efficacy or toxicity. This tion oxygenases are mainly found in enzymes, for example the CYP2 should be considered when multiple the endoplasmic reticulum of the family, are polymorphic (having drugs are simultaneously prescribed liver. The monooxygenase function more than one variant of the gene). and/or when over-the-counter drugs of CYP450 involves a number of Although the CYP isozymes gener- or neutraceuticals are concomitantly steps but the end reaction is the trans- ally have similar functional proper- administered with prescription fer of one oxygen atom to the sub- ties, each one is different and has a drugs. strate (R) that has a site for oxidation distinct role. This polymorphism When several enzymes metabo- as shown below (4). forms a basis for interindividual dif- lize a drug, e.g. propranolol (8), ad- ferences in the efficacy of drug treat- ministration of an enzyme inhibitor NADPH + H+ + O2 + R-H CYP450 NADP+ + H2O + R-OH ment, side effects of drugs and the will not have a great effect since the19 Current Separations 19:1 (2000)
  • 4. T2 Ultra extensive metabolism canKnown inhibitors Enzyme Inducers Inhibitors cause therapeutic failure due to re-and inducers ofCYP isozymes duced bioavailability or lack of acti- CYP1A2 Cigarette Smoke, Phenobarbital, Enoxacin, Ciprofloxacin, Ritonavir, Carbamazepine, Grepafloxacin, vation of the drug whereas poor Charbroiled Foods, Vegetables, Fluvoxamine, Fluoxetine, metabolism can lead to drug toxicity Omeprazole Nefazodone and sometimes death. For optimal CYP2A6 Barbiturates drug therapy, the prescribing physi- cian should have the knowledge of CYP2C9 Rifampin, Carbamazepine, Amiodarone, Fluvastatin, the genetic makeup of the CYP en- Ethanol, Phenytoin Fluvoxamine, Fluoxetine, Fluconazole, Miconazole, zymes in the patient. Metronidazole, Ritonavir, Sulfamethoxazole Outcome of drug metabolism CYP2C19 Rifampin Fluvoxamine, Fluoxetine, Ticlopidine, Ritonavir Various possibilities of the outcome of drug metabolism are illustrated in CYP2D6 Pregnancy Quinidine, Fluoxetine, F4. Paroxetine, Sertraline, Cytochrome P450 reactions Thioridazine, Cimetidine, make substrates more hydrophilic Diphenhydramine, for easy elimination through the kid- Haloperidol, Ticlopidine (Ticlid), Ritonavir neys. Although most often this re- sults in inactivation of the drug, CYP2E1 Ethanol, Isoniazid, Ritonavir Cimetidine, Watercress some compounds form active meta- CYP3A4/5 Carbamazepine, Dexamethasone, Ketoconazole, Itraconazole, bolites. These active metabolites can Rifapentine, Prednisone, Growth Erythromycin, Grapefruit enhance, modify, or inhibit the desir- Hormone, Rifampin, Phenobarbital, Juice, Fluvoxamine, able activity of the drug. Sometimes Phenytoin,Troglitazone Fluoxetine, Diltiazem, Verapamil, Clarithromycin, the active metabolite initiates the Omeprazole), Ritonavir, pharmacological activity. This func- Indinavir tion is used in designing pro drugs. Pro drugs are defined as therapeutic agents that are inactive but are trans-F4 formed into the active form by enzy- Toxicity Toxic MetaboliteEnd results of drug matic reactions. This is very usefulmetabolism Altered Activity when the active form is unstable or Active Metabolite poorly water soluble, making the Enhanced DRUG Activity formulation a challenge. Following Inactive Metabolite Loss of oral administration, the hypotensive Activity drug Enalapril maleate (Vasotec) un- Reversible Metabolite Prolonged dergoes ethyl ester hydrolysis to Activity form enalaprilate, which is the active drug has an alternate pathway. T2 tered to different individuals. Ge- drug. shows some inducers and inhibitors netic polymorphism of CYP450 en- Some drugs have very little of the CYP isozymes. zymes characterize the general therapeutic potential but form a more There is a wide variation in the population into three groups: pharmacologically active metabo- expression, activity and concentra- lite. For example codeine itself has tions of different isozymes among a) Extensive metabolizers (EM): very low analgesic activity. It forms individuals, species and ethnic normal population. morphine, the more active form groups. The expression or the activ- b) Poor metabolizers (PM): Indi- when it is metabolized by CYP2D6. ity of these enzymes is influenced by viduals who inherit two inactive al- Poor metabolizers of CYP2D6 or pa- factors such as species specificity, leles (alternative forms of the gene) tients who are taking CYP2D6 in- genetic polymorphism, gender- hor- showing complete absence of en- hibitors, therefore, do not experience monal control, age, disease and envi- zyme activity. the analgesic property of codeine. ronmental inducers (caffeine, c) Ultra extensive metabolizers In some cases the metabolite ex- cigarette smoke). The variability as- (UEM): Individuals with one com- hibits the same pharmacological ac- sociated with the CYP450 enzymes mon allele and one amplified allele tivity as the parent and is less toxic in each individual results in marked showing enhanced enzyme expres- than the parent. One such example is differences in response when the sion. the antihistamine drug fexofenadine same drug and the dose is adminis- (Allegra) which is a metabolite ofwww.currentseparations.com 20
  • 5. terfenedine (Seldane). Seldane was trials later. Age, hormonal control traceuticals and herbal medicines withdrawn from the market due to its (gender, pregnancy), genetic poly- like St. John’s Wort, Gingko Biloba, fatal interactions with erythromycin morphism, disease state, are all inter- the possibility increases consider- and ketoconazole in some patients nal facto rs that affect the ably for drug interactions to occur. when concomitantly administered. metabolism. Infants for example, While there are many examples lack Phase II enzymes whereas eld- Influence of Drug Metabolism where both parent and the metabolite erly patients have diminished meta- on Drug Development have the same pharmacological ac- bolism and excretion due to the tivity, some metabolites will show aging process. Although there is no In drug development it is important different pharmacological activity evidence of clinically relevant gen- to have information on the enzymes from the parent. This may lead to the der differences in metabolism of hu- responsible for the metabolism of a discovery of a new drug. Loxapine is mans, there have been studies drug candidate as early as possible in an antipsychotic drug that undergoes showing the effect of rat sex hor- the design phase. Knowledge of the extensive metabolism. The N-de- mones on bioavailability. Liver dis- metabolic pathways, metabolite sta- methylated metabolite, amoxapine eases such as hepatitis, liver cancer, bility, toxicity and the specific however has anti depressant activity or cirrhosis impair drug metabolism isozymes involved in the metabolism and is prescribed for that indication. either due to the decreased number are all important information in the Metabolism can also result in of functional hepatocytes or to the drug development process and in toxic metabolites. Formation of re- altered NADPH/NAD ratio in the planning human clinical studies. The active metabolic intermediates is one liver. If the drug is cleared only by rate of metabolism affects the oral of the causes for drug toxicity. Oxi- the liver the impaired metabolism bioavailability and clearance in hu- dation to electrophilic intermediates can result in drug overdose. mans and preclinical species. As dis- or reduction to nucleophilic radicals Genetic or hereditary factors are cu ssed b efo re, polymorphic that can attack DNA or RNA and the most significant factor in drug enzymes will lead to high interindi- induce carcinogenicity are two ma- metabolism (10). Genetic differ- vidual variability and potential for jor reactions by which toxicity is ex- ences among individuals or ethnic DDIs. Genetic information is used to erted. Although many leads are groups can lead to an excessive or predict the response of individual pa- abandoned early on in drug discov- prolonged therapeutic effect or toxic tients and patient populations to ery stage due to the toxic metabolite overdose. For example, the enzyme drugs and to tailor drug selection and formation, presence of a toxic meta- CYP2D6 which metabolizes a large dosage to fit the individual’s genetic bolite does not always implies toxic- number of drugs has 16 alleles. The constitution. Metabolite profiles are ity in a given drug candidate since activity of this enzyme varies widely important for designing prodrugs there are other factors that can make among ethnic groups (11). About 1% and pharmacologically active meta- the metabolite toxic or non-toxic. of Arabics, 30% Chinese and 7-10% bolites and for selecting the right Presence of a toxic metabolite how- Caucasians are poor metabolizers of animal species for toxicology stud- ever raises a red flag, which must be CYP2D6 drugs. Another example is ies. Structural modification of the extensively examined in animal tox- CYP2C19, which contributes to the drug candidate can alter the metabo- icity studies. metabolism of anxiolytics (e.g. di- lism. Highly hydrophilic or highly Some drugs are metabolized re- azepam). About 14-22% Asians and lipophilic compounds are not suit- versibly. For example, sulindac, a 3-6% Caucasians are poor metabo- able because they result in poor nonsteroidal anti-inflammatory drug lizers of CYP2C19. Elevated plasma bioavailability and very slow or very is reversibly metabolized to sulindac drug levels in these populations after fast excretion rates. In these in- sulphide which has anti-inflamma- drug administration can increase the stances replacing an active group tory and analgesic properties and is sedative effect of the drug. with another non-reactive group in irreversibly metabolized to sulindac Environmental factors such as the compound can achieve greater sulphone which has been suggested diet, smoking, alcohol consumption metabolic stability. For example, re- to possess antiproliferative effects and concomitant drug therapy also placing a methyl group by a t-butyl against tumors (9). influence the outcome of drug meta- group can prevent demethylation. bolism. Cigarette smoke produces Similarly, oxidation of aromatic Factors Affecting Drug poly aromatic hydrocarbons (PAH) rings can be prevented by substitut- Metabolism which in duce CYP1A2 (12). ing them with stronger electron with- CYP1A2 metabolizes the PAHs to d rawing groups (e.g. CF3). There are marked differences in drug carcinogens responsible for lung and Information obtained from pre-clini- metabolism across species. Select- colon cancer. Grapefruit juice is a cal drug metabolism studies can be ing a species that closely represents good example of dietary constituent fed back to the design team to intro- the human is very crucial in drug that inhibits CYP3A4 (13). With the duce functional groups which will discovery and in designing clinical new boom in consumption of neu- alter the physical properties to make21 Current Separations 19:1 (2000)
  • 6. T3 tify the metabolites and the sites at Microsomal incubations are mostInformation that can be In vitro studies can which metabolism occur. A variety often used to obtain information onobtained from in vitro give information on: of hepatic in vitro systems differing Phase I reactions. One disadvantagestudies. • Metabolite stability in biological intricacy are now com- is that the information is not com- • Metabolite profile mercially available for metabolism plete as from the cellular systems. • Metabolite identification studies. Most widely used systems • Interspecies comparisons are discussed below in detail. Isolated Hepatocytes • Toxicology species selection • CYP induction/inhibition Cell cultures or cell suspensions can Expressed Enzymes • Drug/Drug interaction studies • CYP isoform identification be used to study multiple aspects of • Phase II enzyme studies Advances in molecular biology have drug metabolism, drug transport enabled the identification and char- across cell membranes, cytotoxicity the compounds more metabolically acterization of a large number of in- and enzyme induction in an environ- stable. Thus final selection of a suc- dividual CYP genes. Specific cDNA ment where enzymes and co-factors cessful drug lead depends im- sequences for particular CYP are present in normal physiological mensely on the drug metabolism isozymes have been cloned and ex- concentrations and cellular integrity studies. pressed heterologously. These ex- is maintained. Hepatocytes are used pressed enzymes including human to study both Phase I and Phase II Pre-clinical Drug enzymes are now commercially reactions. Cells can be either primary Metabolism Studies available as pure systems. Since the or permanent cell cultures. Primary conditions of reactions such as con- cell lines are most often used for drug It is important to know how the drug centrations of enzyme, substrate and metabolism studies because perma- is eliminated early in the drug devel- co-factor can be carefully controlled, nent cell lines possess very little or opment process. If elimination is enzyme systems have become a no enzyme activity. Primary cells are mainly by metabolism, then the me- powerful tool to study drug metabo- isolated from fresh liver tissue and tabolic pathways and products need lism. This system is very useful in the can be used immediately after isola- to be understood. Knowing the tox- study of kinetics, specificity and the tion or culture for long-term studies. icity of a drug and its metabolites mechanism of the enzyme reaction. However, cultured or cryopreserved before entering human clinical trials However, folding or the posttransla- cells lose the P450 activity rapidly is essential to avoid failures later on tional modifications and enzyme ac- with time (14). Also, hepatocytes in the process. Both in vitro and ani- tivity of the expressed enzyme may cannot be frozen and thawed or be mal in vivo studies are done in the differ from the native enzyme. prepared from previously frozen pre-clinical stage. liver. Therefore, there is a great need Most promising compounds are Microsomes for research in improving cryopre- selected from in vitro studies and servation technology and stabiliza- their pharmacokinetic parameters Microsomes can be prepared easily tion of P450 activity in primary are obtained in two animal species, from frozen liver tissues. They con- cultures. With the increased avail- commonly in rat and dog in the in tain most of the oxidative drug me- ability of fresh human tissues from vivo animal studies. This article will tabolizing enzymes. Their easy various commercial and non-profit focus on the in vitro studies and in- preparation and good long-term sta- institutions, human hepatocytes formation obtained from them. bility at -80 °C make microsomes the have become the most widely used most frequently used in vitro system and preferred in vitro system. In Vitro Studies in drug metabolism studies. Mi- crosomes are isolated from liver cells Tissue Slices The in vitro studies during pre-clini- by disrupting the cellular contents cal screening are low-throughput and centrifugation at 100,000 ✕ g. While tissue slices have been used systems. Primary in vitro metabolic Liver microsomes can be manipu- from other organs like brain, heart, systems used in drug metabolism in- lated by induction and inhibition to and kidney, liver is the most com- volve hepatic enzymes or tissue vary the activity or the levels of the monly used tissue type for drug me- preparations. Information (T3) ob- isozymes. The ability to phenotype tabolism experiments. Tissue slices tained by incubating a test drug with microsomes greatly increases the have certain advantages over other these systems can be used as feed- utility of this system in the identifi- systems. With intact cell-cell junc- back to design safer and more meta- cation of specific isozymes responsi- tions, normal hepatic cellular archi- bolically stable drugs. Compounds ble. Metabolic information such as tecture is retained in the tissue. Since can be ranked according to the meta- metabolic profiles, stability, metabo- they contain the complete comple- bolic stabilities. Mass spectrometry lite identification and kinetics can be ment of drug metabolizing enzymes is used as a qualitative tool to iden- obtained from microsomal systems. with all the cofactors present in rele-www.currentseparations.com 22
  • 7. T4 6. H. Shih, G. V. Pickwell, D. K. Guenette, B. Bilir, L. C. Quattrochi,Comparison of System Advantages Disadvantages Future Needs Hum. Exp. Toxicol . 18 (1999)in vitro systems. 95-105 Expressed Pure system Single system Integration with other 7. O. V. Olesen, K. Linnet, J Clin Enzymes enzyme systems Psychopharmacol 20 (2000) 35-42. Microsomes Well-used, Long Limited information, 8. Y. Masubuchi, S. Hosokawa, T. term storage at Need cofactors Horie, T. Suzuki, S. Ohmori, M. -80°C,Well- Kitada, and S. Narimatsu, Drug characterized Metab. Disposition, 22 (1994) 909-915. Isolated cells Integrated cellular Short life time, Increased availability 9. N. M. Davies, M. S. Watson Clin. (e.g. Hepatocytes) system Limited enzyme of human cells, Pharmacokinet. 32 (1997) 437-459 stability Better preservation 10. J. van der Weide, L. S. Steijns Ann Slices Easy to prepare, Limited medium Greater availability Clin Biochem 36 (1999) 722-729. Cellular integrity penetration, of human tissues, 11. H. K. Kroemer, M. Eichelbaum Life maintained Short-term viability Cryopreservation Sciences, 26 (1995) 2285-2298. 12. S. Zevin, N. L. Benowitz Clin Pharmacokinet 36 (1999) 425-438. vant concentrations, complete infor- FDA guidelines suggest first using in 13. D. G. Bailey, J. Malcolm, O. Arnold, mation on the metabolism reactions vitro studies to assess the effect of J. D. Spence Br J Clin Pharmacol 46 (1998) 101-110. can be obtained. Liver slices can be drugs on metabolic pathways and if 14. J. G. Hengstler, D. Utesch, P . easily and rapidly produced. In addi- the results indicate possible DDIs, to Steinberg, K. L .Platt, B. Diener, M. tion, liver slices are not exposed to follow up with in vivo assays (15). Ringel, N. Swales, T. Fischer, K. proteolytic enzymes that can destroy The eventual goal of the in vitro Biefang, M. Gerl, T. Bottger, F. Oesch Drug Metab Rev 32 (2000) important membrane receptors of studies is to predict the in vivo out- 81-118. the cell. Although liver slices are in- come in humans. Utility of human in 15. Guidance for Industry, In Vivo creasingly used now in drug metabo- vitro models to predict drug-drug in- Drug Metabolism/Drug Interaction lism studies, they have certain teraction potential and pharmacoki- Studies -Study Design, Data Analysis, and Recommendations disadvantages. One drawback is the netic variability has been for Dosing and Labeling, Food and inadequate penetration of the me- demonstrated successfully for the Drug Administration, November dium. Liver slices cannot be cryopre- anti psychotic drug, olanzapine (16). 1999. served and they have a limited useful Methodology of scaling in vitro data 16. S. A. Wrighton, B. J. Ring Drug Metab. Rev. 31 (1999) 15-28. experimental period. to predict in vivo outcome, or in vi- 17. J. B. Houston Biochemical Some advantages and limitations tro-in vivo correlation, is expanding Pharmacology 47 (1994) of these in vitro systems are summa- due to the increasing availability of 1469-1479. rized in T4. human in vitro systems (17). Part II With greater availability in hu- of this article will discuss the signifi- man tissues and recombinant en- cance of in vitro enzyme kinetics zymes it is now possible to predict data in the evaluation of in vivo phar- potential DDIs before clinical trials. macokinetic data. Identifying the major metabolic pathways of the drug and its metabo- References lites and exploring the effect of the test drug on the metabolism of other 1. N. Watari, Y. Sugiyama, N. Kaneniwa, M. Hiura. J. drugs and vice versa are two major Pharmacokinetics and goals of the in vitro studies. In vitro Biopharmaceutics, 16 (1988) studies also could help to decide that 279-301. a particular drug is not a substrate for 2. S. A. Wrighton, B. J. Ring, M. VandenBranden, Toxicol. Pathol. 23 certain pathways. This reduces or (1995) 199-208. eliminates the need to study the pos- 3. P J. Murphy, Ed, Year 2000 . sible inhibitory effects of that drug Calendar on History of Drug on other drugs metabolized by that Metabolism, Bioanalytical Systems and ISSX, W. Lafayette, 1999. pathway. 4. M. Spatzenegger, W. Jaeger, Drug Despite the progress in the in Metab. Rev. 27 (1995) 397-417. vitro assays, the general consensus is 5. D. W. Nebert, M. Adenisk, M. J. that in vitro models are too simplistic Coon, R. W. Estabrook, F. J. to substitute for in vivo studies. In Gonzalez, F. P Guengerich, I. C. . Gunsalus, E. F. Johnson, B. vitro studies are best suited to deter- Kemper, W. Levin, I. R. Phillips, R. mine the types of clinical trials Sato, M. R. Waterman, DNA 6 needed to assess potential DDIs. (1987) 1-1123 Current Separations 19:1 (2000)