1. 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
prema@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. These
Drug development
process (IND: in vitro systems include mi-
Investigational New
DISCOVERY crosomes, hepatocytes or tissue
Le
Drug, NDA: New Drug
ad
Application) 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 vitro
17 Current Separations 19:1 (2000)

2. F2 drug as schematically shown in F3 is
Oral Intestines
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-
Schematic representation m Metabolites
ery of the synthetic azo-dye Pron-
bolis
of 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 elimination
www.currentseparations.com 18

3. 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,
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 the
19 Current Separations 19:1 (2000)

4. T2 Ultra extensive metabolism can
Known inhibitors Enzyme Inducers Inhibitors cause therapeutic failure due to re-
and inducers of
CYP 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 Metabolite
End results of drug matic reactions. This is very useful
metabolism 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 of
www.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 make
21 Current Separations 19:1 (2000)

6. T3 tify the metabolites and the sites at Microsomal incubations are most
Information that can be In vitro studies can which metabolism occur. A variety often used to obtain information on
obtained from in vitro give information on: of hepatic in vitro systems differing Phase I reactions. One disadvantage
studies.
• 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
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23 Current Separations 19:1 (2000)