Section I. Basic Principles

Chapter 1. Introduction

Definitions

Pharmacology can be defined as the study of substances that interact with living systems through

chemical processes, especially by binding to regulatory molecules and activating or inhibiting

normal body processes. These substances may be chemicals administered to achieve a beneficial

therapeutic effect on some process within the patient or for their toxic effects on regulatory

processes in parasites infecting the patient. Such deliberate therapeutic applications may be

considered the proper role of medical pharmacology, which is often defined as the science of

substances used to prevent, diagnose, and treat disease. Toxicology is that branch of pharmacology

which deals with the undesirable effects of chemicals on living systems, from individual cells to

complex ecosystems.

History of Pharmacology

Prehistoric people undoubtedly recognized the beneficial or toxic effects of many plant and animal

materials. The earliest written records from China and from Egypt list remedies of many types,

including a few still recognized today as useful drugs. Most, however, were worthless or actually

harmful. In the 2500 years or so preceding the modern era there were sporadic attempts to introduce

rational methods into medicine, but none were successful owing to the dominance of systems of

thought that purported to explain all of biology and disease without the need for experimentation

and observation. These schools promulgated bizarre notions such as the idea that disease was

caused by excesses of bile or blood in the body, that wounds could be healed by applying a salve to

the weapon that caused the wound, and so on.

Around the end of the 17th century, reliance on observation and experimentation began to replace

theorizing in medicine, following the example of the physical sciences. As the value of these

methods in the study of disease became clear, physicians in Great Britain and on the Continent

began to apply them to the effects of traditional drugs used in their own practices. Thus, materia

medica—the science of drug preparation and the medical use of drugs—began to develop as the

precursor to pharmacology. However, any understanding of the mechanisms of action of drugs was

prevented by the absence of methods for purifying active agents from the crude materials that were

available and—even more—by the lack of methods for testing hypotheses about the nature of drug

actions.

In the late 18th and early 19th centuries, François Magendie and later his student Claude Bernard

began to develop the methods of experimental animal physiology and pharmacology. Advances in

chemistry and the further development of physiology in the 18th, 19th, and early 20th centuries laid

the foundation needed for understanding how drugs work at the organ and tissue levels.

Paradoxically, real advances in basic pharmacology during this time were accompanied by an

outburst of unscientific promotion by manufacturers and marketers of worthless "patent medicines."

It was not until the concepts of rational therapeutics, especially that of the controlled clinical trial,

were reintroduced into medicine—about 50 years ago—that it became possible to accurately

evaluate therapeutic claims.

About 50 years ago, there also began a major expansion of research efforts in all areas of biology.

As new concepts and new techniques were introduced, information accumulated about drug action

and the biologic substrate of that action, the receptor. During the last half-century, many

fundamentally new drug groups and new members of old groups were introduced. The last 3

decades have seen an even more rapid growth of information and understanding of the molecular

basis for drug action. The molecular mechanisms of action of many drugs have now been identified,

and numerous receptors have been isolated, structurally characterized, and cloned. In fact, the use of

receptor identification methods (described in Chapter 2: Drug Receptors & Pharmacodynamics) has

led to the discovery of many orphan receptors—receptors for which no ligand has been discovered

and whose function can only be surmised. Studies of the local molecular environment of receptors

have shown that receptors and effectors do not function in isolation—they are strongly influenced

by companion regulatory proteins. Decoding of the genomes of many species—from bacteria to

humans—has led to the recognition of unsuspected relationships between receptor families.

Pharmacogenomics—the relation of the individual's genetic makeup to his or her response to

specific drugs—is close to becoming a practical area of therapy (see Pharmacology & Genetics).

Much of that progress is summarized in this resource.

The extension of scientific principles into everyday therapeutics is still going on, though the

medication-consuming public, unfortunately, is still exposed to vast amounts of inaccurate,

incomplete, or unscientific information regarding the pharmacologic effects of chemicals. This has

resulted in the faddish use of innumerable expensive, ineffective, and sometimes harmful remedies

and the growth of a huge "alternative health care" industry. Conversely, lack of understanding of

basic scientific principles in biology and statistics and the absence of critical thinking about public

health issues has led to rejection of medical science by a segment of the public and a common

tendency to assume that all adverse drug effects are the result of malpractice. Two general

principles that the student should always remember are, first, that all substances can under certain

circumstances be toxic; and second, that all therapies promoted as health-enhancing should meet the

same standards of evidence of efficacy and safety, ie, there should be no artificial separation

between scientific medicine and "alternative" or "complementary" medicine.

Pharmacology & Genetics

During the last 5 years, the genomes of humans, mice, and many other organisms have been

decoded in considerable detail. This has opened the door to a remarkable range of new approaches

to research and treatment. It has been known for centuries that certain diseases are inherited, and we

now understand that individuals with such diseases have a heritable abnormality in their DNA. It is

now possible in the case of some inherited diseases to define exactly which DNA base pairs are

anomalous and in which chromosome they appear. In a small number of animal models of such

diseases, it has been possible to correct the abnormality by "gene therapy," ie, insertion of an

appropriate "healthy" gene into somatic cells. Human somatic cell gene therapy has been attempted,

but the technical difficulties are great.

Studies of a newly discovered receptor or endogenous ligand are often confounded by incomplete

knowledge of the exact role of that receptor or ligand. One of the most powerful of the new genetic

techniques is the ability to breed animals (usually mice) in which the gene for the receptor or its

endogenous ligand has been "knocked out," ie, mutated so that the gene product is absent or

nonfunctional. Homozygous "knockout" mice will usually have complete suppression of that

function, while heterozygous animals will usually have partial suppression. Observation of the

behavior, biochemistry, and physiology of the knockout mice will often define the role of the

missing gene product very clearly. When the products of a particular gene are so essential that even

heterozygotes do not survive to birth, it is sometimes possible to breed "knockdown" versions with

only limited suppression of function. Conversely, "knockin" mice have been bred that overexpress

certain receptors of interest.

Some patients respond to certain drugs with greater than usual sensitivity. (Such variations are

discussed in Chapter 4: Drug Biotransformation.) It is now clear that such increased sensitivity is

often due to a very small genetic modification that results in decreased activity of a particular

enzyme responsible for eliminating that drug. Pharmacogenomics (or pharmacogenetics) is the

study of the genetic variations that cause individual differences in drug response. Future clinicians

may screen every patient for a variety of such differences before prescribing a drug.

The Nature of Drugs

In the most general sense, a drug may be defined as any substance that brings about a change in

biologic function through its chemical actions. In the great majority of cases, the drug molecule

interacts with a specific molecule in the biologic system that plays a regulatory role. This molecule

is called a receptor. The nature of receptors is discussed more fully in Chapter 2: Drug Receptors &

Pharmacodynamics. In a very small number of cases, drugs known as chemical antagonists may

interact directly with other drugs, while a few other drugs (eg, osmotic agents) interact almost

exclusively with water molecules. Drugs may be synthesized within the body (eg, hormones) or

may be chemicals not synthesized in the body, ie, xenobiotics (from Gr xenos "stranger"). Poisons

are drugs. Toxins are usually defined as poisons of biologic origin, ie, synthesized by plants or

animals, in contrast to inorganic poisons such as lead and arsenic.

In order to interact chemically with its receptor, a drug molecule must have the appropriate size,

electrical charge, shape, and atomic composition. Furthermore, a drug is often administered at a

location distant from its intended site of action, eg, a pill given orally to relieve a headache.

Therefore, a useful drug must have the necessary properties to be transported from its site of

administration to its site of action. Finally, a practical drug should be inactivated or excreted from

the body at a reasonable rate so that its actions will be of appropriate duration.

The Physical Nature of Drugs

Drugs may be solid at room temperature (eg, aspirin, atropine), liquid (eg, nicotine, ethanol), or

gaseous (eg, nitrous oxide). These factors often determine the best route of administration. For

example, some liquid drugs are easily vaporized and can be inhaled in that form, eg, halothane,

amyl nitrite. The most common routes of administration are listed in Table 3–3. The various classes

of organic compounds—carbohydrates, proteins, lipids, and their constituents—are all represented

in pharmacology. Many drugs are weak acids or bases. This fact has important implications for the

way they are handled by the body, because pH differences in the various compartments of the body

may alter the degree of ionization of such drugs (see below).

Drug Size

The molecular size of drugs varies from very small (lithium ion, MW 7) to very large (eg, alteplase

[t-PA], a protein of MW 59,050). However, the vast majority of drugs have molecular weights

between 100 and 1000. The lower limit of this narrow range is probably set by the requirements for

specificity of action. In order to have a good "fit" to only one type of receptor, a drug molecule

must be sufficiently unique in shape, charge, etc, to prevent its binding to other receptors. To

achieve such selective binding, it appears that a molecule should in most cases be at least 100 MW

units in size. The upper limit in molecular weight is determined primarily by the requirement that

drugs be able to move within the body (eg, from site of administration to site of action). Drugs

much larger than MW 1000 will not diffuse readily between compartments of the body (see

Permeation, below). Therefore, very large drugs (usually proteins) must be administered directly

into the compartment where they have their effect. In the case of alteplase, a clot-dissolving

enzyme, the drug is administered directly into the vascular compartment by intravenous infusion.

Drug Reactivity and Drug-Receptor Bonds

Drugs interact with receptors by means of chemical forces or bonds. These are of three major types:

covalent, electrostatic, and hydrophobic. Covalent bonds are very strong and in many cases not

reversible under biologic conditions. Thus, the covalent bond formed between the activated form of

phenoxybenzamine and the receptor for norepinephrine (which results in blockade of the receptor)

is not readily broken. The blocking effect of phenoxybenzamine lasts long after the free drug has

disappeared from the bloodstream and is reversed only by the synthesis of new receptors, a

process that takes about 48 hours. Other examples of highly reactive, covalent bond-forming drugs

are the DNA-alkylating agents used in cancer chemotherapy to disrupt cell division in the neoplastic

tissue.

Electrostatic bonding is much more common than covalent bonding in drug-receptor interactions.

Electrostatic bonds vary from relatively strong linkages between permanently charged ionic

molecules to weaker hydrogen bonds and very weak induced dipole interactions such as van der

Waals forces and similar phenomena. Electrostatic bonds are weaker than covalent bonds.

Hydrophobic bonds are usually quite weak and are probably important in the interactions of highly

lipid-soluble drugs with the lipids of cell membranes and perhaps in the interaction of drugs with

the internal walls of receptor "pockets."

The specific nature of a particular drug-receptor bond is of less practical importance than the fact

that drugs which bind through weak bonds to their receptors are generally more selective than drugs

which bind through very strong bonds. This is because weak bonds require a very precise fit of the

drug to its receptor if an interaction is to occur. Only a few receptor types are likely to provide such

a precise fit for a particular drug structure. Thus, if we wished to design a highly selective short-

acting drug for a particular receptor, we would avoid highly reactive molecules that form covalent

bonds and instead choose molecules that form weaker bonds.

A few substances that are almost completely inert in the chemical sense nevertheless have

significant pharmacologic effects. For example, xenon, an "inert gas," has anesthetic effects at

elevated pressures.

Drug Shape

The shape of a drug molecule must be such as to permit binding to its receptor site. Optimally, the

drug's shape is complementary to that of the receptor site in the same way that a key is

complementary to a lock. Furthermore, the phenomenon of chirality (stereoisomerism) is so

common in biology that more than half of all useful drugs are chiral molecules, ie, they exist as

enantiomeric pairs. Drugs with two asymmetric centers have four diastereomers, eg, ephedrine, a

sympathomimetic drug. In the great majority of cases, one of these enantiomers will be much more

potent than its mirror image enantiomer, reflecting a better fit to the receptor molecule. For

example, the (S)(+) enantiomer of methacholine, a parasympathomimetic drug, is over 250 times

more potent than the (R)(–) enantiomer. If one imagines the receptor site to be like a glove into

which the drug molecule must fit to bring about its effect, it is clear why a "left-oriented" drug will

be more effective in binding to a left-hand receptor than will its "right-oriented" enantiomer.

The more active enantiomer at one type of receptor site may not be more active at another type, eg,

a receptor type that may be responsible for some unwanted effect. For example, carvedilol, a drug

that interacts with adrenoceptors, has a single chiral center and thus two enantiomers (Table 1–1).

One of these enantiomers, the (S)(–) isomer, is a potent -receptor blocker. The (R)(+) isomer is

100-fold weaker at the receptor. However, the isomers are approximately equipotent as -receptor

blockers. Ketamine is an intravenous anesthetic. The (+) enantiomer is a more potent anesthetic and

is less toxic than the (–) enantiomer. Unfortunately, the drug is still used as the racemic mixture.

Table 1–1. Dissociation Constants (K

) of the Enantiomers and Racemate of Carvedilol.

Form of

Carvedilol

Inverse of Affinity for Receptors

, nmol/L)

Inverse of Affinity for Receptors

, nmol/L)

R(+) enantiomer 14 45

S(–) enantiomer 16 0.4

R,S(+/–)

enantiomers

11 0.9

Note: The K

is the concentration for 50% saturation of the receptors and is inversely proportionate

to the affinity of the drug for the receptors.

Data from Ruffolo RR et al: The pharmacology of carvedilol. Eur J Pharmacol 1990;38:S82.

Finally, because enzymes are usually stereoselective, one drug enantiomer is often more susceptible

than the other to drug-metabolizing enzymes. As a result, the duration of action of one enantiomer

may be quite different from that of the other.

Unfortunately, most studies of clinical efficacy and drug elimination in humans have been carried

out with racemic mixtures of drugs rather than with the separate enantiomers. At present, only about

45% of the chiral drugs used clinically are marketed as the active isomer—the rest are available

only as racemic mixtures. As a result, many patients are receiving drug doses of which 50% or more

is either inactive or actively toxic. However, there is increasing interest—at both the scientific and

the regulatory levels—in making more chiral drugs available as their active enantiomers.

Rational Drug Design

Rational design of drugs implies the ability to predict the appropriate molecular structure of a drug

on the basis of information about its biologic receptor. Until recently, no receptor was known in

sufficient detail to permit such drug design. Instead, drugs were developed through random testing

of chemicals or modification of drugs already known to have some effect (see Chapter 5: Basic &

Clinical Evaluation of New Drugs). However, during the past 3 decades, many receptors have been

isolated and characterized. A few drugs now in use were developed through molecular design based

on a knowledge of the three-dimensional structure of the receptor site. Computer programs are now

available that can iteratively optimize drug structures to fit known receptors. As more becomes

known about receptor structure, rational drug design will become more feasible.

Receptor Nomenclature

The spectacular success of newer, more efficient ways to identify and characterize receptors (see

Chapter 2: Drug Receptors & Pharmacodynamics, How Are New Receptors Discovered?) has

resulted in a variety of differing systems for naming them. This in turn has led to a number of

suggestions regarding more rational methods of naming them. The interested reader is referred for

details to the efforts of the International Union of Pharmacology (IUPHAR) Committee on Receptor

Nomenclature and Drug Classification (reported in various issues of Pharmacological Reviews)

and to the annual Receptor and Ion Channel Nomenclature Supplements published as special issues

by the journal Trends in Pharmacological Sciences (TIPS). The chapters in this book mainly use

these sources for naming receptors.

Drug-Body Interactions

The interactions between a drug and the body are conveniently divided into two classes. The actions

of the drug on the body are termed pharmacodynamic processes; the principles of

pharmacodynamics are presented in greater detail in Chapter 2: Drug Receptors &

Pharmacodynamics. These properties determine the group in which the drug is classified and often

play the major role in deciding whether that group is appropriate therapy for a particular symptom

or disease. The actions of the body on the drug are called pharmacokinetic processes and are

described in Chapters 3 and 4. Pharmacokinetic processes govern the absorption, distribution, and

elimination of drugs and are of great practical importance in the choice and administration of a

particular drug for a particular patient, eg, one with impaired renal function. The following

paragraphs provide a brief introduction to pharmacodynamics and pharmacokinetics.

Pharmacodynamic Principles

As noted above, most drugs must bind to a receptor to bring about an effect. However, at the

molecular level, drug binding is only the first in what is often a complex sequence of steps.

Types of Drug-Receptor Interactions

Agonist drugs bind to and activate the receptor in some fashion, which directly or indirectly brings

about the effect. Some receptors incorporate effector machinery in the same molecule, so that drug

binding brings about the effect directly, eg, opening of an ion channel or activation of enzyme

activity. Other receptors are linked through one or more intervening coupling molecules to a

separate effector molecule. The several types of drug-receptor-effector coupling systems are

discussed in Chapter 2: Drug Receptors & Pharmacodynamics. Pharmacologic antagonist drugs, by

binding to a receptor, prevent binding by other molecules. For example, acetylcholine receptor

blockers such as atropine are antagonists because they prevent access of acetylcholine and similar

agonist drugs to the acetylcholine receptor and they stabilize the receptor in its inactive state. These

agents reduce the effects of acetylcholine and similar drugs in the body.

"Agonists" That Inhibit Their Binding Molecules and Partial Agonists

Some drugs mimic agonist drugs by inhibiting the molecules responsible for terminating the action

of an endogenous agonist. For example, acetylcholinesterase inhibitors, by slowing the destruction

of endogenous acetylcholine, cause cholinomimetic effects that closely resemble the actions of

cholinoceptor agonist molecules even though cholinesterase inhibitors do not—or only incidentally

do—bind to cholinoceptors (see Chapter 7: Cholinoceptor-Activating & Cholinesterase-Inhibiting

Drugs). Other drugs bind to receptors and activate them but do not evoke as great a response as so-

called full agonists. Thus, pindolol, a adrenoceptor "partial agonist," may act as either an agonist

(if no full agonist is present) or as an antagonist (if a full agonist such as isoproterenol is present).

(See Chapter 2: Drug Receptors & Pharmacodynamics.)

Duration of Drug Action

Termination of drug action at the receptor level results from one of several processes. In some

cases, the effect lasts only as long as the drug occupies the receptor, so that dissociation of drug

from the receptor automatically terminates the effect. In many cases, however, the action may

persist after the drug has dissociated, because, for example, some coupling molecule is still present

in activated form. In the case of drugs that bind covalently to the receptor, the effect may persist

until the drug-receptor complex is destroyed and new receptors are synthesized, as described

previously for phenoxybenzamine. Finally, many receptor-effector systems incorporate

desensitization mechanisms for preventing excessive activation when agonist molecules continue to

be present for long periods. See Chapter 2: Drug Receptors & Pharmacodynamics for additional

details.

Receptors and Inert Binding Sites

To function as a receptor, an endogenous molecule must first be selective in choosing ligands (drug

molecules) to bind; and second, it must change its function upon binding in such a way that the

function of the biologic system (cell, tissue, etc) is altered. The first characteristic is required to

avoid constant activation of the receptor by promiscuous binding of many different ligands. The

second characteristic is clearly necessary if the ligand is to cause a pharmacologic effect. The body

contains many molecules that are capable of binding drugs, however, and not all of these

endogenous molecules are regulatory molecules. Binding of a drug to a nonregulatory molecule

such as plasma albumin will result in no detectable change in the function of the biologic system, so

this endogenous molecule can be called an inert binding site. Such binding is not completely

without significance, however, since it affects the distribution of drug within the body and will

determine the amount of free drug in the circulation. Both of these factors are of pharmacokinetic

importance (see below and Chapter 3: Pharmacokinetics & Pharmacodynamics: Rational Dosing &

the Time Course of Drug Action).

Pharmacokinetic Principles

In practical therapeutics, a drug should be able to reach its intended site of action after

administration by some convenient route. In some cases, a chemical that is readily absorbed and

distributed is administered and then converted to the active drug by biologic processes—inside the

body. Such a chemical is called a prodrug. In only a few situations is it possible to directly apply a

drug to its target tissue, eg, by topical application of an anti-inflammatory agent to inflamed skin or

mucous membrane. Most often, a drug is administered into one body compartment, eg, the gut, and

must move to its site of action in another compartment, eg, the brain. This requires that the drug be

absorbed into the blood from its site of administration and distributed to its site of action,

permeating through the various barriers that separate these compartments. For a drug given orally

to produce an effect in the central nervous system, these barriers include the tissues that comprise

the wall of the intestine, the walls of the capillaries that perfuse the gut, and the "blood-brain

barrier," the walls of the capillaries that perfuse the brain. Finally, after bringing about its effect, a

drug should be eliminated at a reasonable rate by metabolic inactivation, by excretion from the

body, or by a combination of these processes.

Permeation

Drug permeation proceeds by four primary mechanisms. Passive diffusion in an aqueous or lipid

medium is common, but active processes play a role in the movement of many drugs, especially

those whose molecules are too large to diffuse readily.

Aqueous Diffusion

Aqueous diffusion occurs within the larger aqueous compartments of the body (interstitial space,

cytosol, etc) and across epithelial membrane tight junctions and the endothelial lining of blood

vessels through aqueous pores that—in some tissues—permit the passage of molecules as large as

MW 20,000–30,000.

The capillaries of the brain, the testes, and some other tissues are characterized by absence of the

pores that permit aqueous diffusion of many drug molecules into the tissue. They may also contain

high concentrations of drug export pumps (MDR pumps; see text). These tissues are therefore

"protected" or "sanctuary" sites from many circulating drugs.

Aqueous diffusion of drug molecules is usually driven by the concentration gradient of the

permeating drug, a downhill movement described by Fick's law (see below). Drug molecules that

are bound to large plasma proteins (eg, albumin) will not permeate these aqueous pores. If the drug

is charged, its flux is also influenced by electrical fields (eg, the membrane potential and—in parts

of the nephron—the transtubular potential).

Lipid Diffusion

Lipid diffusion is the most important limiting factor for drug permeation because of the large

number of lipid barriers that separate the compartments of the body. Because these lipid barriers

separate aqueous compartments, the lipid:aqueous partition coefficient of a drug determines how

readily the molecule moves between aqueous and lipid media. In the case of weak acids and weak

bases (which gain or lose electrical charge-bearing protons, depending on the pH), the ability to

move from aqueous to lipid or vice versa varies with the pH of the medium, because charged

molecules attract water molecules. The ratio of lipid-soluble form to water-soluble form for a weak

acid or weak base is expressed by the Henderson-Hasselbalch equation (see below).

Special Carriers

Special carrier molecules exist for certain substances that are important for cell function and too

large or too insoluble in lipid to diffuse passively through membranes, eg, peptides, amino acids,

glucose. These carriers bring about movement by active transport or facilitated diffusion and, unlike

passive diffusion, are saturable and inhibitable. Because many drugs are or resemble such naturally

occurring peptides, amino acids, or sugars, they can use these carriers to cross membranes.

Many cells also contain less selective membrane carriers that are specialized for expelling foreign

molecules, eg, the P-glycoprotein or multidrug-resistance type 1 (MDR1) transporter found in

the brain, testes, and other tissues, and in some drug-resistant neoplastic cells. A similar transport

molecule, the multidrug resistance-associated protein-type 2 (MRP2) transporter, plays an

important role in excretion of some drugs or their metabolites into urine and bile.

Endocytosis and Exocytosis

A few substances are so large or impermeant that they can enter cells only by endocytosis, the

process by which the substance is engulfed by the cell membrane and carried into the cell by

pinching off of the newly formed vesicle inside the membrane. The substance can then be released

inside the cytosol by breakdown of the vesicle membrane. This process is responsible for the

transport of vitamin B

, complexed with a binding protein (intrinsic factor), across the wall of the

gut into the blood. Similarly, iron is transported into hemoglobin-synthesizing red blood cell

precursors in association with the protein transferrin. Specific receptors for the transport proteins

must be present for this process to work. The reverse process (exocytosis) is responsible for the

secretion of many substances from cells. For example, many neurotransmitter substances are stored

in membrane-bound vesicles in nerve endings to protect them from metabolic destruction in the

cytoplasm. Appropriate activation of the nerve ending causes fusion of the storage vesicle with the

cell membrane and expulsion of its contents into the extracellular space (see Chapter 6: Introduction

to Autonomic Pharmacology).

Fick's Law of Diffusion

The passive flux of molecules down a concentration gradient is given by Fick's law:

where C

is the higher concentration, C

is the lower concentration, area is the area across which

diffusion is occurring, permeability coefficient is a measure of the mobility of the drug molecules in

the medium of the diffusion path, and thickness is the thickness (length) of the diffusion path. In the

case of lipid diffusion, the lipid:aqueous partition coefficient is a major determinant of mobility of

the drug, since it determines how readily the drug enters the lipid membrane from the aqueous

medium.

Ionization of Weak Acids and Weak Bases; the Henderson-Hasselbalch Equation

The electrostatic charge of an ionized molecule attracts water dipoles and results in a polar,

relatively water-soluble and lipid-insoluble complex. Since lipid diffusion depends on relatively

high lipid solubility, ionization of drugs may markedly reduce their ability to permeate membranes.

A very large fraction of the drugs in use are weak acids or weak bases (Table 1–2). For drugs, a

weak acid is best defined as a neutral molecule that can reversibly dissociate into an anion (a

negatively charged molecule) and a proton (a hydrogen ion). For example, aspirin dissociates as

follows:

Table 1–2. Ionization Constants of Some Common Drugs.

Drug pK

Weak acids

Acetaminophen 9.5

Acetazolamide 7.2

Ampicillin 2.5

Aspirin 3.5

Chlorothiazide 6.8, 9.4

Chlorpropamide 5.0

Ciprofloxacin 6.09, 8.74

Cromolyn 2.0

Ethacrynic acid 2.5

Furosemide 3.9

Ibuprofen 4.4, 5.2

Levodopa 2.3

Methotrexate 4.8

Methyldopa 2.2, 9.2

Penicillamine 1.8

Pentobarbital 8.1

Phenobarbital 7.4

Phenytoin 8.3

Propylthiouracil 8.3

Salicylic acid 3.0

Sulfadiazine 6.5

Sulfapyridine 8.4

Theophylline 8.8

Tolbutamide 5.3

Warfarin 5.0

Weak bases

Albuterol (salbutamol) 9.3

Allopurinol 9.4, 12.3

Alprenolol 9.6

Amiloride 8.7

Amiodarone 6.56

Amphetamine 9.8

Atropine 9.7

Bupivacaine 8.1

Chlordiazepoxide 4.6

Chloroquine 10.8, 8.4

Chlorpheniramine 9.2

Chlorpromazine 9.3

Clonidine 8.3

Cocaine 8.5

Codeine 8.2

Cyclizine 8.2

Desipramine 10.2

Diazepam 3

Dihydrocodeine 3

Diphenhydramine 8.8

Diphenoxylate 7.1

Ephedrine 9.6

Epinephrine 8.7

Ergotamine 6.3

Fluphenazine 8.0, 3.9

Guanethidine 11.4, 8.3

Hydralazine 7.1

Imipramine 9.5

Isoproterenol 8.6

Kanamycin 7.2

Lidocaine 7.9

Metaraminol 8.6

Methadone 8.4

Methamphetamine 10.0

Methyldopa 10.6

Metoprolol 9.8

Morphine 7.9

Nicotine 7.9, 3.1

Norepinephrine 8.6

Pentazocine 7.9

Phenylephrine 9.8

Physostigmine 7.9, 1.8

Pilocarpine 6.9, 1.4

Pindolol 8.6

Procainamide 9.2

Procaine 9.0

Promazine 9.4

Promethazine 9.1

Propranolol 9.4

Pseudoephedrine 9.8

Pyrimethamine 7.0

Quinidine 8.5, 4.4

Scopolamine 8.1

Strychnine 8.0, 2.3

Terbutaline 10.1

Thioridazine 9.5

Tolazoline 10.6

A drug that is a weak base can be defined as a neutral molecule that can form a cation (a positively

charged molecule) by combining with a proton. For example, pyrimethamine, an antimalarial drug,

undergoes the following association-dissociation process:

Note that the protonated form of a weak acid is the neutral, more lipid-soluble form, whereas the

unprotonated form of a weak base is the neutral form. The law of mass action requires that these

reactions move to the left in an acid environment (low pH, excess protons available) and to the right

in an alkaline environment. The Henderson-Hasselbalch equation relates the ratio of protonated to

unprotonated weak acid or weak base to the molecule's pK

and the pH of the medium as follows:

This equation applies to both acidic and basic drugs. Inspection confirms that the lower the pH

relative to the pK

, the greater will be the fraction of drug in the protonated form. Because the

uncharged form is the more lipid-soluble, more of a weak acid will be in the lipid-soluble form at

acid pH, while more of a basic drug will be in the lipid-soluble form at alkaline pH.

An application of this principle is in the manipulation of drug excretion by the kidney. Almost all

drugs are filtered at the glomerulus. If a drug is in a lipid-soluble form during its passage down the

renal tubule, a significant fraction will be reabsorbed by simple passive diffusion. If the goal is to

accelerate excretion of the drug, it is important to prevent its reabsorption from the tubule. This can

often be accomplished by adjusting urine pH to make certain that most of the drug is in the ionized

state, as shown in Figure 1–1. As a result of this pH partitioning effect, the drug will be "trapped" in

the urine. Thus, weak acids are usually excreted faster in alkaline urine; weak bases are usually

excreted faster in acidic urine. Other body fluids in which pH differences from blood pH may cause

trapping or reabsorption are the contents of the stomach and small intestine; breast milk; aqueous

humor; and vaginal and prostatic secretions (Table 1–3).

Figure 1–1.

Trapping of a weak base (pyrimethamine) in the urine when the urine is more acidic than the

blood. In the hypothetical case illustrated, the diffusible uncharged form of the drug has

equilibrated across the membrane but the total concentration (charged plus uncharged) in the urine

is almost eight times higher than in the blood.

Table 1–3. Body Fluids with Potential for Drug "Trapping" Through the pH-Partitioning

Phenomenon.

Body Fluid Range

of pH

Total Fluid: Blood

Concentration Ratios for

Sulfadiazine (acid, pK

6.5)

Total Fluid: Blood Concentration

Ratios for Pyrimethamine (base,

7.0)

Urine 5.0–8.0 0.12–4.65 72.24–0.79

Breast milk 6.4–7.6

0.2–1.77 3.56–0.89

Jejunum,

ileum

contents

7.5–8.0

1.23–3.54 0.94–0.79

Stomach

contents

1.92–

2.59

0.11

85,993–18,386

Prostatic

secretions

6.45–

7.4

0.21 3.25–1.0

Vaginal

secretions

3.4–4.2

0.11

2848–452

Body fluid protonated-to-unprotonated drug ratios were calculated using each of the pH extremes

cited; a blood pH of 7.4 was used for blood:drug ratio. For example, the steady-state urine:blood

ratio for sulfadiazine is 0.12 at a urine pH of 5.0; this ratio is 4.65 at a urine pH of 8.0. Thus,

sulfadiazine is much more effectively trapped and excreted in alkaline urine.

Lentner C (editor): Geigy Scientific Tables, vol 1, 8th ed. Ciba Geigy, 1981.

Bowman WC, Rand MJ: Textbook of Pharmacology, 2nd ed. Blackwell, 1980.

Insignificant change in ratios over the physiologic pH range.

As suggested by Table 1–2, a large number of drugs are weak bases. Most of these bases are amine-

containing molecules. The nitrogen of a neutral amine has three atoms associated with it plus a pair

of unshared electrons—see the display below. The three atoms may consist of one carbon

(designated "R") and two hydrogens (a primary amine), two carbons and one hydrogen (a

secondary amine), or three carbon atoms (a tertiary amine). Each of these three forms may

reversibly bind a proton with the unshared electrons. Some drugs have a fourth carbon-nitrogen

bond; these are quaternary amines. However, the quaternary amine is permanently charged and has

no unshared electrons with which to reversibly bind a proton. Therefore, primary, secondary, and

tertiary amines may undergo reversible protonation and vary their lipid solubility with pH, but

quaternary amines are always in the poorly lipid-soluble charged form.

Drug Groups

To learn each pertinent fact about each of the many hundreds of drugs mentioned in this book

would be an impractical goal and, fortunately, is in any case unnecessary. Almost all of the several

thousand drugs currently available can be arranged in about 70 groups. Many of the drugs within

each group are very similar in pharmacodynamic actions and often in their pharmacokinetic

properties as well. For most groups, one or more prototype drugs can be identified that typify the

most important characteristics of the group. This permits classification of other important drugs in

the group as variants of the prototype, so that only the prototype must be learned in detail and, for

the remaining drugs, only the differences from the prototype.

Sources of Information

Students who wish to review the field of pharmacology in preparation for an examination are

referred to Pharmacology: Examination and Board Review, by Trevor, Katzung, and Masters

(McGraw-Hill, 2002) or USMLE Road Map: Pharmacology, by Katzung and Trevor (McGraw-Hill,

2003).

The references at the end of each chapter in this book were selected to provide information specific

to those chapters.

Specific questions relating to basic or clinical research are best answered by resort to the general

pharmacology and clinical specialty serials. For the student and the physician, three periodicals can

be recommended as especially useful sources of current information about drugs: The New England

Journal of Medicine, which publishes much original drug-related clinical research as well as

frequent reviews of topics in pharmacology; The Medical Letter on Drugs and Therapeutics, which

publishes brief critical reviews of new and old therapies, mostly pharmacologic; and Drugs, which

publishes extensive reviews of drugs and drug groups.

Other sources of information pertinent to the USA should be mentioned as well. The "package

insert" is a summary of information the manufacturer is required to place in the prescription sales

package; Physicians' Desk Reference (PDR) is a compendium of package inserts published annually

with supplements twice a year; Facts and Comparisons is a more complete loose-leaf drug

information service with monthly updates; and the USP DI (vol 1, Drug Information for the Health

Care Professional) is a large drug compendium with monthly updates that is now published on the

Internet by the Micromedex Corporation. The package insert consists of a brief description of the

pharmacology of the product. While this brochure contains much practical information, it is also

used as a means of shifting liability for untoward drug reactions from the manufacturer onto the

practitioner. Therefore, the manufacturer typically lists every toxic effect ever reported, no matter

how rare. A useful and objective handbook that presents information on drug toxicity and

interactions is Drug Interactions. Finally, the FDA has an Internet World Wide Web site that carries

news regarding recent drug approvals, withdrawals, warnings, etc. It can be reached using a

personal computer equipped with Internet browser software at http://www.fda.gov.

The following addresses are provided for the convenience of readers wishing to obtain any of the

publications mentioned above:

Drug Interactions

Lea & Febiger

600 Washington Square

Philadelphia, PA 19106

Facts and Comparisons

111 West Port Plaza, Suite 300

St. Louis, MO 63146

Pharmacology: Examination & Board Review, 6th ed

McGraw-Hill Companies, Inc

2 Penn Plaza 12th Floor

New York, NY 10121-2298

USMLE Road Map: Pharmacology

McGraw-Hill Companies, Inc

2 Penn Plaza 12th Floor

New York, NY 10121-2298

The Medical Letter on Drugs and Therapeutics

56 Harrison Street

New Rochelle, NY 10801

The New England Journal of Medicine

10 Shattuck Street

Boston, MA 02115

Physicians' Desk Reference

Box 2017

Mahopac, NY 10541

United States Pharmacopeia Dispensing Information

Micromedex, Inc.

6200 S. Syracuse Way, Suite 300

Englewood, CO 80111

Chapter 2. Drug Receptors & Pharmacodynamics

Drug Receptors & Pharmacodynamics: Introduction

Therapeutic and toxic effects of drugs result from their interactions with molecules in the patient.

Most drugs act by associating with specific macromolecules in ways that alter the macromolecules'

biochemical or biophysical activities. This idea, more than a century old, is embodied in the term

receptor: the component of a cell or organism that interacts with a drug and initiates the chain of

biochemical events leading to the drug's observed effects.

Receptors have become the central focus of investigation of drug effects and their mechanisms of

action (pharmacodynamics). The receptor concept, extended to endocrinology, immunology, and

molecular biology, has proved essential for explaining many aspects of biologic regulation. Many

drug receptors have been isolated and characterized in detail, thus opening the way to precise

understanding of the molecular basis of drug action.

The receptor concept has important practical consequences for the development of drugs and for

arriving at therapeutic decisions in clinical practice. These consequences form the basis for

understanding the actions and clinical uses of drugs described in almost every chapter of this book.

They may be briefly summarized as follows:

(1) Receptors largely determine the quantitative relations between dose or concentration of

drug and pharmacologic effects. The receptor's affinity for binding a drug determines the

concentration of drug required to form a significant number of drug-receptor complexes, and the

total number of receptors may limit the maximal effect a drug may produce.

(2) Receptors are responsible for selectivity of drug action. The molecular size, shape, and

electrical charge of a drug determine whether—and with what affinity—it will bind to a particular

receptor among the vast array of chemically different binding sites available in a cell, tissue, or

patient. Accordingly, changes in the chemical structure of a drug can dramatically increase or

decrease a new drug's affinities for different classes of receptors, with resulting alterations in

therapeutic and toxic effects.

(3) Receptors mediate the actions of both pharmacologic agonists and antagonists. Some drugs

and many natural ligands, such as hormones and neurotransmitters, regulate the function of receptor

macromolecules as agonists; ie, they activate the receptor to signal as a direct result of binding to it.

Other drugs function as pharmacologic antagonists; ie, they bind to receptors but do not activate

generation of a signal; consequently, they interfere with the ability of an agonist to activate the

receptor. Thus, the effect of a so-called "pure" antagonist on a cell or in a patient depends entirely

on its preventing the binding of agonist molecules and blocking their biologic actions. Some of the

most useful drugs in clinical medicine are pharmacologic antagonists.

Macromolecular Nature of Drug Receptors

Most receptors are proteins, presumably because the structures of polypeptides provide both the

necessary diversity and the necessary specificity of shape and electrical charge. The section How

Are New Receptors Discovered? describes some of the methods by which receptors are discovered

and defined.

The best-characterized drug receptors are regulatory proteins, which mediate the actions of

endogenous chemical signals such as neurotransmitters, autacoids, and hormones. This class of

receptors mediates the effects of many of the most useful therapeutic agents. The molecular

structures and biochemical mechanisms of these regulatory receptors are described in a later section

entitled Signaling Mechanisms & Drug Action.

Other classes of proteins that have been clearly identified as drug receptors include enzymes, which

may be inhibited (or, less commonly, activated) by binding a drug (eg, dihydrofolate reductase, the

receptor for the antineoplastic drug methotrexate); transport proteins (eg, Na

ATPase, the

membrane receptor for cardioactive digitalis glycosides); and structural pro-teins (eg, tubulin, the

receptor for colchicine, an anti-inflammatory agent).

This chapter deals with three aspects of drug receptor function, presented in increasing order of

complexity: (1) Receptors as determinants of the quantitative relation between the concentration of

a drug and the pharmacologic response. (2) Receptors as regulatory proteins and components of

chemical signaling mechanisms that provide targets for important drugs. (3) Receptors as key

determinants of the therapeutic and toxic effects of drugs in patients.

How Are New Receptors Discovered?

Because today's new receptor sets the stage for tomorrow's new drug, it is important to know how

new receptors are discovered. Receptor discovery often begins by studying the relations between

structures and activities of a group of drugs on some conveniently measured response. Binding of

radioactive ligands defines the molar abundance and binding affinities of the putative receptor and

provides an assay to aid in its biochemical purification.

Analysis of the pure receptor protein identifies the number of its subunits, its size, and (sometimes)

provides a clue to how it works (eg, agonist-stimulated autophosphorylation on tyrosine residues,

seen with receptors for insulin and many growth factors). These classic steps in receptor

identification serve as a warming-up exercise for molecular cloning of the segment of DNA that

encodes the receptor. Receptors within a specific class or subclass generally contain highly

conserved regions of similar or identical amino acid (and therefore DNA) sequence. This has led to

an entirely different approach toward identifying receptors by sequence homology to already known

(cloned) receptors.

Cloning of new receptors by sequence homology has identified a number of subtypes of known

receptor classes, such as -adrenoceptors and serotonin receptors, the diversity of which was only

partially anticipated from pharmacologic studies. This approach has also led to the identification of

receptors whose existence was not anticipated from pharmacologic studies. These putative

receptors, identified only by their similarity to other known receptors, are termed orphan receptors

until their native ligands are identified. Identifying such receptors and their ligands is of great

interest because this process may elucidate entirely new signaling pathways and therapeutic targets.

Relation between Drug Concentration & Response

The relation between dose of a drug and the clinically observed response may be complex. In

carefully controlled in vitro systems, however, the relation between concentration of a drug and its

effect is often simple and can be described with mathematical precision. This idealized relation

underlies the more complex relations between dose and effect that occur when drugs are given to

patients.

Concentration-Effect Curves & Receptor Binding of Agonists

Even in intact animals or patients, responses to low doses of a drug usually increase in direct

proportion to dose. As doses increase, however, the response increment diminishes; finally, doses

may be reached at which no further increase in response can be achieved. In idealized or in vitro

systems, the relation between drug concentration and effect is described by a hyperbolic curve

(Figure 2–1 A) according to the following equation:

where E is the effect observed at concentration C, E

max

is the maximal response that can be

produced by the drug, and EC

is the concentration of drug that produces 50% of maximal effect.

Figure 2–1.

Relations between drug concentration and drug effect (panel A) or receptor-bound drug (panel B).

The drug concentrations at which effect or receptor occupancy is half-maximal are denoted EC

and K

, respectively.

This hyperbolic relation resembles the mass action law, which predicts association between two

molecules of a given affinity. This resemblance suggests that drug agonists act by binding to

("occupying") a distinct class of biologic molecules with a characteristic affinity for the drug

receptor. Radioactive receptor ligands have been used to confirm this occupancy assumption in

many drug-receptor systems. In these systems, drug bound to receptors (B) relates to the

concentration of free (unbound) drug (C) as depicted in Figure 2–1 B and as described by an

analogous equation:

in which B

max

indicates the total concentration of receptor sites (ie, sites bound to the drug at

infinitely high concentrations of free drug). K

(the equilibrium dissociation constant) represents

the concentration of free drug at which half-maximal binding is observed. This constant

characterizes the receptor's affinity for binding the drug in a reciprocal fashion: If the K

is low,

binding affinity is high, and vice versa. The EC

and K

may be identical, but need not be, as

discussed below. Dose-response data is often presented as a plot of the drug effect (ordinate) against

the logarithm of the dose or concentration (abscissa). This mathematical maneuver transforms the

hyperbolic curve of Figure 2–1 into a sigmoid curve with a linear midportion (eg, Figure 2–2). This

expands the scale of the concentration axis at low concentrations (where the effect is changing

rapidly) and compresses it at high concentrations (where the effect is changing slowly), but has no

special biologic or pharmacologic significance.

Figure 2–2.

Logarithmic transformation of the dose axis and experimental demonstration of spare receptors,

using different concentrations of an irreversible antagonist. Curve A shows agonist response in the

absence of antagonist. After treatment with a low concentration of antagonist (curve B), the curve

is shifted to the right; maximal responsiveness is preserved, however, because the remaining

available receptors are still in excess of the number required. In curve C, produced after treatment

with a larger concentration of antagonist, the available receptors are no longer "spare"; instead,

they are just sufficient to mediate an undiminished maximal response. Still higher concentrations

of antagonist (curves D and E) reduce the number of available receptors to the point that maximal

response is diminished. The apparent EC

of the agonist in curves D and E may approximate the

that characterizes the binding affinity of the agonist for the receptor.

Receptor-Effector Coupling & Spare Receptors

When a receptor is occupied by an agonist, the resulting conformational change is only the first of

many steps usually required to produce a pharmacologic response. The transduction process

between occupancy of receptors and drug response is often termed coupling. The relative efficiency

of occupancy-response coupling is partially determined by the initial conformational change in the

receptor—thus, the effects of full agonists can be considered more efficiently coupled to receptor

occupancy than can the effects of partial agonists, as described below. Coupling efficiency is also

determined by the biochemical events that transduce receptor occupancy into cellular response.

High efficiency of receptor-effector interaction may also be envisioned as the result of spare

receptors. Receptors are said to be "spare" for a given pharmacologic response when the maximal

response can be elicited by an agonist at a concentration that does not result in occupancy of the full

complement of available receptors. Spare receptors are not qualitatively different from nonspare

receptors. They are not hidden or unavailable, and when they are occupied they can be coupled to

response. Experimentally, spare receptors may be demonstrated by using irreversible antagonists to

prevent binding of agonist to a proportion of available receptors and showing that high

concentrations of agonist can still produce an undiminished maximal response (Figure 2–2). Thus, a

maximal inotropic response of heart muscle to catecholamines can be elicited even under conditions

where 90% of the -adrenoceptors are occupied by a quasi-irreversible antagonist. Accordingly,

myocardial cells are said to contain a large proportion of spare -adrenoceptors.

How can we account for the phenomenon of spare receptors? In a few cases, the biochemical

mechanism is understood, such as for drugs that act on some regulatory receptors. In this situation,

the effect of receptor activation—eg, binding of guanosine triphosphate (GTP) by an intermediate—

may greatly outlast the agonist-receptor interaction (see the following section on G Proteins &

Second Messengers). In such a case, the "spareness" of receptors is temporal in that the response

initiated by an individual ligand-receptor binding event persists longer than the binding event itself.

In other cases, where the biochemical mechanism is not understood, we imagine that the receptors

might be spare in number. If the concentration or amount of a cellular component other than the

receptor limits the coupling of receptor occupancy to response, then a maximal response can occur

without occupancy of all receptors. This concept helps explain how the sensitivity of a cell or tissue

to a particular concentration of agonist depends not only on the affinity of the receptor for binding

the agonist (characterized by the K

) but also on the degree of spareness—the total number of

receptors present compared to the number actually needed to elicit a maximal biologic response.

The K

of the agonist-receptor interaction determines what fraction (B/B

max

) of total receptors will

be occupied at a given free concentration (C) of agonist regardless of the receptor concentration:

Imagine a responding cell with four receptors and four effectors. Here the number of effectors does

not limit the maximal response, and the receptors are not spare in number. Consequently, an agonist

present at a concentration equal to the K

will occupy 50% of the receptors, and half of the

effectors will be activated, producing a half-maximal response (ie, two receptors stimulate two

effectors). Now imagine that the number of receptors increases 10-fold to 40 receptors but that the

total number of effectors remains constant. Most of the receptors are now spare in number. As a

result, a much lower concentration of agonist suffices to occupy two of the 40 receptors (5% of the

receptors), and this same low concentration of agonist is able to elicit a half-maximal response (two

of four effectors activated). Thus, it is possible to change the sensitivity of tissues with spare

receptors by changing the receptor concentration.

Competitive & Irreversible Antagonists

Receptor antagonists bind to receptors but do not activate them. In general, the effects of these

antagonists result from preventing agonists (other drugs or endogenous regulatory molecules) from

binding to and activating receptors. Such antagonists are divided into two classes depending on

whether or not they reversibly compete with agonists for binding to receptors.

In the presence of a fixed concentration of agonist, increasing concentrations of a competitive

antagonist progressively inhibit the agonist response; high antagonist concentrations prevent

response completely. Conversely, sufficiently high concentrations of agonist can completely

surmount the effect of a given concentration of the antagonist; ie, the E

max

for the agonist remains

the same for any fixed concentration of antagonist (Figure 2–3 A). Because the antagonism is

competitive, the presence of antagonist increases the agonist concentration required for a given

degree of response, and so the agonist concentration-effect curve is shifted to the right.

Figure 2–3.

Changes in agonist concentration-effect curves produced by a competitive antagonist (panel A) or

by an irreversible antagonist (panel B). In the presence of a competitive antagonist, higher

concentrations of agonist are required to produce a given effect; thus the agonist concentration (C')

required for a given effect in the presence of concentration [I] of an antagonist is shifted to the

right, as shown. High agonist concentrations can overcome inhibition by a competitive antagonist.

This is not the case with an irreversible antagonist, which reduces the maximal effect the agonist

can achieve, although it may not change its EC

The concentration (C') of an agonist required to produce a given effect in the presence of a fixed

concentration ([I]) of competitive antagonist is greater than the agonist concentration (C) required

to produce the same effect in the absence of the antagonist. The ratio of these two agonist

concentrations (the "dose ratio") is related to the dissociation constant (K

) of the antagonist by the

Schild equation:

Pharmacologists often use this relation to determine the K

of a competitive antagonist. Even

without knowledge of the relationship between agonist occupancy of the receptor and response, the

can be determined simply and accurately. As shown in Figure 2–3, concentration response

curves are obtained in the presence and in the absence of a fixed concentration of competitive

antagonist; comparison of the agonist concentrations required to produce identical degrees of

pharmacologic effect in the two situations reveals the antagonist's K

. If C' is twice C, for example,

then [I] = K

For the clinician, this mathematical relation has two important therapeutic implications:

(1) The degree of inhibition produced by a competitive antagonist depends on the

concentration of antagonist. Different patients receiving a fixed dose of propranolol, for

example, exhibit a wide range of plasma concentrations, owing to differences in clearance of

the drug. As a result, the effects of a fixed dose of this competitive antagonist of

norepinephrine may vary widely in patients, and the dose must be adjusted accordingly.

(2) Clinical response to a competitive antagonist depends on the concentration of agonist

that is competing for binding to receptors. Here also propranolol provides a useful example:

When this competitive -adrenoceptor antagonist is administered in doses sufficient to block

the effect of basal levels of the neurotransmitter norepinephrine, resting heart rate is

decreased. However, the increase in release of norepinephrine and epinephrine that occurs

with exercise, postural changes, or emotional stress may suffice to overcome competitive

antagonism by propranolol and increase heart rate, and thereby can influence therapeutic

response.

Some receptor antagonists bind to the receptor in an irreversible or nearly irreversible fashion, ie,

not competitive. The antagonist's affinity for the receptor may be so high that for practical purposes,

the receptor is unavailable for binding of agonist. Other antagonists in this class produce

irreversible effects because after binding to the receptor they form covalent bonds with it. After

occupancy of some proportion of receptors by such an antagonist, the number of remaining

unoccupied receptors may be too low for the agonist (even at high concentrations) to elicit a

response comparable to the previous maximal response (Figure 2–3 B). If spare receptors are

present, however, a lower dose of an irreversible antagonist may leave enough receptors unoccupied

to allow achievement of maximum response to agonist, although a higher agonist concentration will

be required (Figures 2–2 B and C; see Receptor-Effector Coupling and Spare Receptors, above).

Therapeutically, irreversible antagonists present distinctive advantages and disadvantages. Once the

irreversible antagonist has occupied the receptor, it need not be present in unbound form to inhibit

agonist responses. Consequently, the duration of action of such an irreversible antagonist is

relatively independent of its own rate of elimination and more dependent on the rate of turnover of

receptor molecules.

Phenoxybenzamine, an irreversible -adrenoceptor antagonist, is used to control the hypertension

caused by catecholamines released from pheochromocytoma, a tumor of the adrenal medulla. If

administration of phenoxybenzamine lowers blood pressure, blockade will be maintained even

when the tumor episodically releases very large amounts of catecholamine. In this case, the ability

to prevent responses to varying and high concentrations of agonist is a therapeutic advantage. If

overdose occurs, however, a real problem may arise. If the -adrenoceptor blockade cannot be

overcome, excess effects of the drug must be antagonized "physiologically," ie, by using a pressor

agent that does not act via receptors.

Partial Agonists

Based on the maximal pharmacologic response that occurs when all receptors are occupied, agonists

can be divided into two classes: partial agonists produce a lower response, at full receptor

occupancy, than do full agonists. Partial agonists produce concentration-effect curves that resemble

those observed with full agonists in the presence of an antagonist that irreversibly blocks some of

the receptor sites (compare Figures 2–2 [curve D] and 2–4 B). It is important to emphasize that the

failure of partial agonists to produce a maximal response is not due to decreased affinity for binding

to receptors. Indeed, a partial agonist's inability to cause a maximal pharmacologic response, even

when present at high concentrations that saturate binding to all receptors, is indicated by the fact

that partial agonists competitively inhibit the responses produced by full agonists (Figure 2–4 C).

Many drugs used clinically as antagonists are in fact weak partial agonists.

Figure 2–4.

Panel A: The percentage of receptor occupancy resulting from full agonist (present at a single

concentration) binding to receptors in the presence of increasing concentrations of a partial

agonist. Because the full agonist (filled squares) and the partial agonist (open squares) compete to

bind to the same receptor sites, when occupancy by the partial agonist increases, binding of the full

agonist decreases. Panel B: When each of the two drugs is used alone and response is measured,

occupancy of all the receptors by the partial agonist produces a lower maximal response than does

similar occupancy by the full agonist. Panel C: Simultaneous treatment with a single concentration

of full agonist and increasing concentrations of the partial agonist produces the response patterns

shown in the bottom panel. The fractional response caused by a single concentration of the full

agonist (filled squares) decreases as increasing concentrations of the partial agonist compete to

bind to the receptor with increasing success; at the same time the portion of the response caused by

the partial agonist (open squares) increases, while the total response—ie, the sum of responses to

the two drugs (filled triangles)—gradually decreases, eventually reaching the value produced by

partial agonist alone (compare panel B).

Other Mechanisms of Drug Antagonism

Not all of the mechanisms of antagonism involve interactions of drugs or endogenous ligands at a

single type of receptor. Indeed, chemical antagonists need not involve a receptor at all. Thus, one

drug may antagonize the actions of a second drug by binding to and inactivating the second drug.

For example, protamine, a protein that is positively charged at physiologic pH, can be used

clinically to counteract the effects of heparin, an anticoagulant that is negatively charged; in this

case, one drug antagonizes the other simply by binding it and making it unavailable for interactions

with proteins involved in formation of a blood clot.

The clinician often uses drugs that take advantage of physiologic antagonism between endogenous

regulatory pathways. For example, several catabolic actions of the glucocorticoid hormones lead to

increased blood sugar, an effect that is physiologically opposed by insulin. Although

glucocorticoids and insulin act on quite distinct receptor-effector systems, the clinician must

sometimes administer insulin to oppose the hyperglycemic effects of glucocorticoid hormone,

whether the latter is elevated by endogenous synthesis (eg, a tumor of the adrenal cortex) or as a

result of glucocorticoid therapy.

In general, use of a drug as a physiologic antagonist produces effects that are less specific and less

easy to control than are the effects of a receptor-specific antagonist. Thus, for example, to treat

bradycardia caused by increased release of acetylcholine from vagus nerve endings, the physician

could use isoproterenol, a -adrenoceptor agonist that increases heart rate by mimicking

sympathetic stimulation of the heart. However, use of this physiologic antagonist would be less

rational—and potentially more dangerous—than would use of a receptor-specific antagonist such as

atropine (a competitive antagonist at the receptors at which acetylcholine slows heart rate).

Signaling Mechanisms & Drug Action

Until now we have considered receptor interactions and drug effects in terms of equations and

concentration-effect curves. We must also understand the molecular mechanisms by which a drug

acts. Such understanding allows us to ask basic questions with important clinical implications:

• Why do some drugs produce effects that persist for minutes, hours, or even days after the

drug is no longer present?

• Why do responses to other drugs diminish rapidly with prolonged or repeated

administration?

• How do cellular mechanisms for amplifying external chemical signals explain the

phenomenon of spare receptors?

• Why do chemically similar drugs often exhibit extraordinary selectivity in their actions?

• Do these mechanisms provide targets for developing new drugs?

Most transmembrane signaling is accomplished by a small number of different molecular

mechanisms. Each type of mechanism has been adapted, through the evolution of distinctive protein

families, to transduce many different signals. These protein families include receptors on the cell

surface and within the cell, as well as enzymes and other components that generate, amplify,

coordinate, and terminate postreceptor signaling by chemical second messengers in the cytoplasm.

This section first discusses the mechanisms for carrying chemical information across the plasma

membrane and then outlines key features of cytoplasmic second messengers.

Five basic mechanisms of transmembrane signaling are well understood (Figure 2–5). Each uses a

different strategy to circumvent the barrier posed by the lipid bilayer of the plasma membrane.

These strategies use (1) a lipid-soluble ligand that crosses the membrane and acts on an intracellular

receptor; (2) a transmembrane receptor protein whose intracellular enzymatic activity is

allosterically regulated by a ligand that binds to a site on the protein's extracellular domain; (3) a

transmembrane receptor that binds and stimulates a protein tyrosine kinase; (4) a ligand-gated

transmembrane ion channel that can be induced to open or close by the binding of a ligand; or (5) a

transmembrane receptor protein that stimulates a GTP-binding signal transducer protein (G protein),

which in turn modulates production of an intracellular second messenger.

Figure 2–5.

Known transmembrane signaling mechanisms: 1: A lipid-soluble chemical signal crosses the

plasma membrane and acts on an intracellular receptor (which may be an enzyme or a regulator of

gene transcription); 2: the signal binds to the extracellular domain of a transmembrane protein,

thereby activating an enzymatic activity of its cytoplasmic domain; 3: the signal binds to the

extracellular domain of a transmembrane receptor bound to a protein tyrosine kinase, which it

activates; 4: the signal binds to and directly regulates the opening of an ion channel; 5: the signal

binds to a cell-surface receptor linked to an effector enzyme by a G protein. (A,C, substrates; B, D,

products; R, receptor; G, G protein; E, effector [enzyme or ion channel]; Y, tyrosine; P,

phosphate.)

While the five established mechanisms do not account for all the chemical signals conveyed across

cell membranes, they do transduce many of the most important signals exploited in

pharmacotherapy.

Intracellular Receptors for Lipid-Soluble Agents

Several biologic signals are sufficiently lipid-soluble to cross the plasma membrane and act on

intracellular receptors. One of these is a gas, nitric oxide (NO), that acts by stimulating an

intracellular enzyme, guanylyl cyclase, which produces cyclic guanosine monophosphate (cGMP).

Signaling via cGMP is described in more detail later in this chapter. Receptors for another class of

ligands—including corticosteroids, mineralocorticoids, sex steroids, vitamin D, and thyroid

hormone—stimulate the transcription of genes in the nucleus by binding to specific DNA sequences

near the gene whose expression is to be regulated. Many of the target DNA sequences (called

response elements) have been identified.

These "gene-active" receptors belong to a protein family that evolved from a common precursor.

Dissection of the receptors by recombinant DNA techniques has provided insights into their

molecular mechanism. For example, binding of glucocorticoid hormone to its normal receptor

protein relieves an inhibitory constraint on the transcription-stimulating activity of the protein.

Figure 2–6 schematically depicts the molecular mechanism of glucocorticoid action: In the absence

of hormone, the receptor is bound to hsp90, a protein that appears to prevent normal folding of

several structural domains of the receptor. Binding of hormone to the ligand-binding domain

triggers release of hsp90. This allows the DNA-binding and transcription-activating domains of the

receptor to fold into their functionally active conformations, so that the activated receptor can

initiate transcription of target genes.

Figure 2–6.

Mechanism of glucocorticoid action. The glucocorticoid receptor polypeptide is schematically

depicted as a protein with three distinct domains. A heat-shock protein, hsp90, binds to the

receptor in the absence of hormone and prevents folding into the active conformation of the

receptor. Binding of a hormone ligand (steroid) causes dissociation of the hsp90 stabilizer and

permits conversion to the active configuration.

The mechanism used by hormones that act by regulating gene expression has two therapeutically

important consequences:

(1) All of these hormones produce their effects after a characteristic lag period of 30 minutes to

several hours—the time required for the synthesis of new proteins. This means that the gene-

active hormones cannot be expected to alter a pathologic state within minutes (eg,

glucocorticoids will not immediately relieve the symptoms of acute bronchial asthma).

(2) The effects of these agents can persist for hours or days after the agonist concentration has

been reduced to zero. The persistence of effect is primarily due to the relatively slow turnover of

most enzymes and proteins, which can remain active in cells for hours or days after they have

been synthesized. Consequently, it means that the beneficial (or toxic) effects of a gene-active

hormone will usually decrease slowly when administration of the hormone is stopped.

Ligand-Regulated Transmembrane Enzymes Including Receptor Tyrosine Kinases

This class of receptor molecules mediates the first steps in signaling by insulin, epidermal growth

factor (EGF), platelet-derived growth factor (PDGF), atrial natriuretic peptide (ANP), transforming

growth factor- (TGF- ), and many other trophic hormones. These receptors are polypeptides

consisting of an extracellular hormone-binding domain and a cytoplasmic enzyme domain, which

may be a protein tyrosine kinase, a serine kinase, or a guanylyl cyclase (Figure 2–7). In all these

receptors, the two domains are connected by a hydrophobic segment of the polypeptide that crosses

the lipid bilayer of the plasma membrane.

Figure 2–7.

Mechanism of activation of the epidermal growth factor (EGF) receptor, a representative receptor

tyrosine kinase. The receptor polypeptide has extracellular and cytoplasmic domains, depicted

above and below the plasma membrane. Upon binding of EGF (circle), the receptor converts from

its inactive monomeric state (left) to an active dimeric state (right), in which two receptor

polypeptides bind noncovalently in the plane of the membrane. The cytoplasmic domains become

phosphorylated (P) on specific tyrosine residues (Y) and their enzymatic activities are activated,

catalyzing phosphorylation of substrate proteins (S).

The receptor tyrosine kinase signaling pathway begins with ligand binding to the receptor's

extracellular domain. The resulting change in receptor conformation causes receptor molecules to

bind to one another, which in turn brings together the tyrosine kinase domains, which become

enzymatically active, and phosphorylate one another as well as additional downstream signaling

proteins. Activated receptors catalyze phosphorylation of tyrosine residues on different target

signaling proteins, thereby allowing a single type of activated receptor to modulate a number of

biochemical processes. Insulin, for example, uses a single class of receptors to trigger increased

uptake of glucose and amino acids and to regulate metabolism of glycogen and triglycerides in the

cell. Similarly, each of the growth factors initiates in its specific target cells a complex program of

cellular events ranging from altered membrane transport of ions and metabolites to changes in the

expression of many genes. At present, a few compounds have been found to produce effects that

may be due to inhibition of tyrosine kinase activities. It is easy to imagine therapeutic uses for

specific inhibitors of growth factor receptors, especially in neoplastic disorders where excessive

growth factor signaling is often observed. For example, a monoclonal antibody (trastuzumab) that

acts as an antagonist of the HER2/neu receptor tyrosine kinase is effective in therapy of human

breast cancers associated with overexpression of this growth factor receptor.

The intensity and duration of action of EGF, PDGF, and other agents that act via receptor tyrosine

kinases are limited by receptor down-regulation. Ligand binding induces accelerated endocytosis of

receptors from the cell surface, followed by the degradation of those receptors (and their bound

ligands). When this process occurs at a rate faster than de novo synthesis of receptors, the total

number of cell-surface receptors is reduced (down-regulated) and the cell's responsiveness to ligand

is correspondingly diminished. A well-understood process by which many tyrosine kinases are

down-regulated is via ligand-induced internalization of receptors followed by trafficking to

lysosomes, where receptors are proteolyzed. EGF causes internalization and subsequent proteolytic

down-regulation after binding to the EGF receptor protein tyrosine kinase; genetic mutations that

interfere with this process of down-regulation cause excessive growth factor–induced cell

proliferation and are associated with an increased susceptibility to certain types of cancer.

Internalization of certain receptor tyrosine kinases, most notably receptors for nerve growth factor,

serves a very different function. Internalized nerve growth factor receptors are not rapidly degraded.

Instead, receptors remain intact and are translocated in endocytic vesicles from the distal axon

(where receptors are activated by nerve growth factor released from the innervated tissue) to the cell

body (where the signal is transduced to transcription factors regulating the expression of genes

controlling cell survival). This process effectively transports a critical survival signal released from

the target tissue over a remarkably long distance—more than 1 meter in certain sensory neurons. A

number of regulators of growth and differentiation, including TGF- , act on another class of

transmembrane receptor enzymes that phosphorylate serine and threonine residues. ANP, an

important regulator of blood volume and vascular tone, acts on a transmembrane receptor whose

intracellular domain, a guanylyl cyclase, generates cGMP (see below). Receptors in both groups,

like the receptor tyrosine kinases, are active in their dimeric forms.

Cytokine Receptors

Cytokine receptors respond to a heterogeneous group of peptide ligands that includes growth

hormone, erythropoietin, several kinds of interferon, and other regulators of growth and

differentiation. These receptors use a mechanism (Figure 2–8) closely resembling that of receptor

tyrosine kinases, except that in this case, the protein tyrosine kinase activity is not intrinsic to the

receptor molecule. Instead, a separate protein tyrosine kinase, from the Janus-kinase (JAK) family,

binds noncovalently to the receptor. As in the case of the EGF-receptor, cytokine receptors dimerize

after they bind the activating ligand, allowing the bound JAKs to become activated and to

phosphorylate tyrosine residues on the receptor. Tyrosine phosphates on the receptor then set in

motion a complex signaling dance by binding another set of proteins, called STATs (signal

transducers and activators of transcription). The bound STATs are themselves phosphorylated by

the JAKs, two STAT molecules dimerize (attaching to one another's tyrosine phosphates), and

finally the STAT/STAT dimer dissociates from the receptor and travels to the nucleus, where it

regulates transcription of specific genes.

Figure 2–8.

Cytokine receptors, like receptor tyrosine kinases, have extracellular and intracellular domains and

form dimers. However, after activation by an appropriate ligand, separate mobile protein tyrosine

kinase molecules (JAK) are activated, resulting in phosphorylation of signal transducers and

activation of transcription (STAT) molecules. STAT dimers then travel to the nucleus, where they

regulate transcription.

Ligand-Gated Channels

Many of the most useful drugs in clinical medicine act by mimicking or blocking the actions of

endogenous ligands that regulate the flow of ions through plasma membrane channels. The natural

ligands include acetylcholine, serotonin, -aminobutyric acid (GABA), and the excitatory amino

acids (eg, glycine, aspartate, and glutamate). All of these agents are synaptic transmitters.

Each of their receptors transmits its signal across the plasma membrane by increasing

transmembrane conductance of the relevant ion and thereby altering the electrical potential across

the membrane. For example, acetylcholine causes the opening of the ion channel in the nicotinic

acetylcholine receptor (AChR), which allows Na

to flow down its concentration gradient into cells,

producing a localized excitatory postsynaptic potential—a depolarization.

The AChR (Figure 2–9) is one of the best-characterized of all cell-surface receptors for hormones

or neurotransmitters. One form of this receptor is a pentamer made up of five polypeptide subunits

(eg, two chains plus one , one , and one chain, all with molecular weights ranging from 43,000

to 50,000). These polypeptides, each of which crosses the lipid bilayer four times, form a cylindric

structure 8 nm in diameter. When acetylcholine binds to sites on the subunits, a conformational

change occurs that results in the transient opening of a central aqueous channel through which

sodium ions penetrate from the extracellular fluid into the cell.

Figure 2–9.

The nicotinic acetylcholine receptor, a ligand-gated ion channel. The receptor molecule is depicted

as embedded in a rectangular piece of plasma membrane, with extracellular fluid above and

cytoplasm below. Composed of five subunits (two , one , one , and one ), the receptor opens a

central transmembrane ion channel when acetylcholine (ACh) binds to sites on the extracellular

domain of its subunits.

The time elapsed between the binding of the agonist to a ligand-gated channel and the cellular

response can often be measured in milliseconds. The rapidity of this signaling mechanism is

crucially important for moment-to-moment transfer of information across synapses. Ligand-gated

ion channels can be regulated by multiple mechanisms, including phosphorylation and

internalization. In the central nervous system, these mechanisms contribute to synaptic plasticity

involved in learning and memory.

G Proteins & Second Messengers

Many extracellular ligands act by increasing the intracellular concentrations of second messengers

such as cyclic adenosine-3',5'-monophosphate (cAMP), calcium ion, or the phosphoinositides

(described below). In most cases they use a transmembrane signaling system with three separate

components. First, the extracellular ligand is specifically detected by a cell-surface receptor. The

receptor in turn triggers the activation of a G protein located on the cytoplasmic face of the plasma

membrane. The activated G protein then changes the activity of an effector element, usually an

enzyme or ion channel. This element then changes the concentration of the intracellular second

messenger. For cAMP, the effector enzyme is adenylyl cyclase, a transmembrane protein that

converts intracellular adenosine triphosphate (ATP) to cAMP. The corresponding G protein, G

stimulates adenylyl cyclase after being activated by hormones and neurotransmitters that act via a

specific receptor (Table 2–1).

Table 2–1. A Partial List of Endogenous Ligands and Their Associated Second Messengers.

Ligand Second Messenger

Adrenocorticotropic hormone cAMP

Acetylcholine (muscarinic receptors) Ca

, phosphoinositides

Angiotensin Ca

, phosphoinositides

Catecholamines (

-adrenoceptors)

, phosphoinositides

Catecholamines ( -adrenoceptors) cAMP

Chorionic gonadotropin cAMP

Follicle-stimulating hormone cAMP

Glucagon cAMP

Histamine (H

receptors)

cAMP

Luteinizing hormone cAMP

Melanocyte-stimulating hormone cAMP

Parathyroid hormone cAMP

Platelet-activating factor Ca

, phosphoinositides

Prostacyclin, prostaglandin E2 cAMP

Serotonin (5-HT

receptors)

cAMP

Serotonin (5-HT

and 5-HT

receptors)

, phosphoinositides

Thyrotropin cAMP

Thyrotropin-releasing hormone Ca

, phosphoinositides

Vasopressin (V

receptors)

, phosphoinositides

Vasopressin (V

receptors) cAMP

Key: cAMP = cyclic adenosine monophosphate.

and other G proteins use a molecular mechanism that involves binding and hydrolysis of GTP

(Figure 2–10). This mechanism allows the transduced signal to be amplified. For example, a

neurotransmitter such as norepinephrine may encounter its membrane receptor for only a few

milliseconds. When the encounter generates a GTP-bound G

molecule, however, the duration of

activation of adenylyl cyclase depends on the longevity of GTP binding to G

rather than on the

receptor's affinity for norepinephrine. Indeed, like other G proteins, GTP-bound G

may remain

active for tens of seconds, enormously amplifying the original signal. This mechanism explains how

signaling by G proteins produces the phenomenon of spare receptors (described above). At low

concentrations of agonist the proportion of agonist-bound receptors may be much less than the

proportion of G proteins in the active (GTP-bound) state; if the proportion of active G proteins

correlates with pharmacologic response, receptors will appear to be spare (ie, a small fraction of

receptors occupied by agonist at any given time will appear to produce a proportionately larger

response).

Figure 2–10.

The guanine nucleotide-dependent activation-inactivation cycle of G proteins. The agonist

activates the receptor (R), which promotes release of GDP from the G protein (G), allowing entry

of GTP into the nucleotide binding site. In its GTP-bound state (G-GTP), the G protein regulates

activity of an effector enzyme or ion channel (E). The signal is terminated by hydrolysis of GTP,

followed by return of the system to the basal unstimulated state. Open arrows denote regulatory

effects. (P

, inorganic phosphate.)

The family of G proteins contains several functionally diverse subfamilies (Table 2–2), each of

which mediates effects of a particular set of receptors to a distinctive group of effectors. Receptors

coupled to G proteins comprise a family of "seven-transmembrane" or "serpentine" receptors, so

called because the receptor polypeptide chain "snakes" across the plasma membrane seven times

(Figure 2–11). Receptors for adrenergic amines, serotonin, acetylcholine (muscarinic but not

nicotinic), many peptide hormones, odorants, and even visual receptors (in retinal rod and cone

cells) all belong to the serpentine family. All were derived from a common evolutionary precursor.

Some serpentine receptors exist as dimers, but it is thought that dimerization is not usually required

for activation.

Table 2–2. G Proteins and Their Receptors and Effectors.

Protein

Receptors for: Effector/Signaling Pathway

-Adrenergic amines, glucagon, histamine,

serotonin, and many other hormones

Adenylyl cyclase cAMP

, G

-Adrenergic amines, acetylcholine

(muscarinic), opioids, serotonin, and many others

Several, including:

Adenylyl cyclase cAMP

Open cardiac K

channels heart

rate

olf

Odorants (olfactory epithelium) Adenylyl cyclase cAMP

Neurotransmitters in brain (not yet specifically

identified)

Not yet clear

Acetylcholine (eg, muscarinic), bombesin,

serotonin (5-HT

), and many others

Phospholipase C IP

diacylglycerol, cytoplasmic Ca

, G

Photons (rhodopsin and color opsins in retinal

rod and cone cells)

cGMP phosphodiesterase cGMP

(phototransduction)

Key: cAMP = cyclic adenosine monophosphate; cGMP = cyclic guanosine monophosphate.

Serpentine receptors transduce signals across the plasma membrane in essentially the same way.

Often the agonist ligand—eg, a catecholamine, acetylcholine, or the photon-activated chromophore

of retinal photoreceptors—is bound in a pocket enclosed by the transmembrane regions of the

receptor (as in Figure 2–11). The resulting change in conformation of these regions is transmitted to

cytoplasmic loops of the receptor, which in turn activate the appropriate G protein by promoting

replacement of GDP by GTP, as described above. Considerable biochemical evidence indicates that

G proteins interact with amino acids in the third cytoplasmic loop of the serpentine receptor

polypeptide (shown by arrows in Figure 2–11). The carboxyl terminal tails of these receptors, also

located in the cytoplasm, can regulate the receptors' ability to interact with G proteins, as described

below.

Figure 2–11.

Transmembrane topology of a typical serpentine receptor. The receptor's amino (N) terminal is

extracellular (above the plane of the membrane), and its carboxyl (C) terminal intracellular. The

terminals are connected by a polypeptide chain that traverses the plane of the membrane seven

times. The hydrophobic transmembrane segments (light color) are designated by roman numerals

(I–VII). The agonist (Ag) approaches the receptor from the extracellular fluid and binds to a site

surrounded by the transmembrane regions of the receptor protein. G proteins (G) interact with

cytoplasmic regions of the receptor, especially with portions of the third cytoplasmic loop between

transmembrane regions V and VI. The receptor's cytoplasmic terminal tail contains numerous

serine and threonine residues whose hydroxyl (–OH) groups can be phosphorylated. This

phosphorylation may be associated with diminished receptor-G protein interaction.

Receptor Regulation

Receptor-mediated responses to drugs and hormonal agonists often desensitize with time (Figure 2–

12, top). After reaching an initial high level, the response (eg, cellular cAMP accumulation, Na

influx, contractility, etc) gradually diminishes over seconds or minutes, even in the continued

presence of the agonist. This desensitization is usually reversible; a second exposure to agonist, if

provided a few minutes after termination of the first exposure, results in a response similar to the

initial response.

Figure 2–12.

Possible mechanism for desensitization of the -adrenoceptor. The upper part of the figure depicts

the response to a -adrenoceptor agonist (ordinate) versus time (abscissa). The break in the time

axis indicates passage of time in the absence of agonist. Temporal duration of exposure to agonist

is indicated by the light-colored bar. The lower part of the figure schematically depicts agonist-

induced phosphorylation (P) by -adrenoceptor kinase ( -adrenergic receptor kinase, ARK) of

carboxyl terminal hydroxyl groups (–OH) of the -adrenoceptor. This phosphorylation induces

binding of a protein, -arrestin ( -arr), which prevents the receptor from interacting with G

Removal of agonist for a short period of time allows dissociation of -arr, removal of phosphate

(Pi) from the receptor by phosphatases (P'ase), and restoration of the receptor's normal

responsiveness to agonist.

Although many kinds of receptors undergo desensitization, the mechanism is in many cases

obscure. A molecular mechanism of desensitization has been worked out in some detail, however,

in the case of the -adrenoceptor (Figure 2–12, bottom). The agonist-induced change in

conformation of the receptor causes it to bind, activate, and serve as a substrate for a specific

kinase, -adrenoceptor kinase (also called ARK). ARK then phosphorylates serine or threonine

residues in the receptor's carboxyl terminal tail. The presence of phosphoserines increases the

receptor's affinity for binding a third protein, -arrestin. Binding of -arrestin to cytoplasmic loops

of the receptor diminishes the receptor's ability to interact with G

, thereby reducing the agonist

response (ie, stimulation of adenylyl cyclase). Upon removal of agonist, however, cellular

phosphatases remove phosphates from the receptor and ARK stops putting them back on, so that

the receptor—and consequently the agonist response—return to normal. This mechanism of

desensitization, which rapidly and reversibly modulates the ability of the receptor to signal to G

protein, turns out to regulate many G protein–coupled receptors. Another important regulatory

process is down-regulation. Down-regulation, which decreases the actual number of receptors

present in the cell or tissue, occurs more slowly than rapid desensitization and is less readily

reversible. This is because down-regulation involves a net degradation of receptors present in the

cell, requiring new receptor biosynthesis for recovery, in contrast to rapid desensitization which

involves reversible phosphorylation of existing receptors. Many G protein–coupled receptors are

down-regulated by undergoing ligand-induced endocytosis and delivery to lysosomes, similar to

down-regulation of protein tyrosine kinases such as the EGF receptor. Down-regulation generally

occurs only after prolonged or repeated exposure of cells to agonist (over hours to days). Brief

periods of agonist exposure (several minutes) can also induce internalization of receptors. In this

case, many receptors, including the -adrenoceptor, do not down-regulate but instead recycle intact

to the plasma membrane. This rapid cycling through endocytic vesicles promotes dephosphorylation

of receptors, increasing the rate at which fully functional receptors are replenished in the plasma

membrane. Thus, depending on the particular receptor and duration of activation, internalization

can mediate quite different effects on receptor signaling and regulation.

Well-Established Second Messengers

Cyclic Adenosine Monophosphate (cAMP)

Acting as an intracellular second messenger, cAMP mediates such hormonal responses as the

mobilization of stored energy (the breakdown of carbohydrates in liver or triglycerides in fat cells

stimulated by -adrenomimetic catecholamines), conservation of water by the kidney (mediated by

vasopressin), Ca

homeostasis (regulated by parathyroid hormone), and increased rate and

contraction force of heart muscle (

-adrenomimetic catecholamines). It also regulates the

production of adrenal and sex steroids (in response to corticotropin or follicle-stimulating

hormone), relaxation of smooth muscle, and many other endocrine and neural processes.

cAMP exerts most of its effects by stimulating cAMP-dependent protein kinases (Figure 2–13).

These kinases are composed of a cAMP-binding regulatory (R) dimer and two catalytic (C) chains.

When cAMP binds to the R dimer, active C chains are released to diffuse through the cytoplasm

and nucleus, where they transfer phosphate from ATP to appropriate substrate proteins, often

enzymes. The specificity of cAMP's regulatory effects resides in the distinct protein substrates of

the kinases that are expressed in different cells. For example, liver is rich in phosphorylase kinase

and glycogen synthase, enzymes whose reciprocal regulation by cAMP-dependent phosphorylation

governs carbohydrate storage and release.

Figure 2–13.

The cAMP second messenger pathway. Key proteins include hormone receptors (Rec), a

stimulatory G protein (G

), catalytic adenylyl cyclase (AC), phosphodiesterases (PDE) that

hydrolyze cAMP, cAMP-dependent kinases, with regulatory (R) and catalytic (C) subunits, protein

substrates (S) of the kinases, and phosphatases (P'ase), which remove phosphates from substrate

proteins. Open arrows denote regulatory effects.

When the hormonal stimulus stops, the intracellular actions of cAMP are terminated by an elaborate

series of enzymes. cAMP-stimulated phosphorylation of enzyme substrates is rapidly reversed by a

diverse group of specific and nonspecific phosphatases. cAMP itself is degraded to 5'-AMP by

several cyclic nucleotide phosphodiesterases (PDE, Figure 2–13). Competitive inhibition of cAMP

degradation is one way caffeine, theophylline, and other methylxanthines produce their effects (see

Chapter 20: Drugs Used in Asthma).

Calcium and Phosphoinositides

Another well-studied second messenger system involves hormonal stimulation of phosphoinositide

hydrolysis (Figure 2–14). Some of the hormones, neurotransmitters, and growth factors that trigger

this pathway (see Table 2–1) bind to receptors linked to G proteins, while others bind to receptor

tyrosine kinases. In all cases, the crucial step is stimulation of a membrane enzyme, phospholipase

C (PLC), which splits a minor phospholipid component of the plasma membrane,

phosphatidylinositol-4,5-bisphosphate (PIP

), into two second messengers, diacylglycerol and

inositol-1,4,5-trisphosphate (IP

or InsP

). Diacylglycerol is confined to the membrane where it

activates a phospholipid- and calcium-sensitive protein kinase called protein kinase C. IP

is water-

soluble and diffuses through the cytoplasm to trigger release of Ca

from internal storage vesicles.

Elevated cytoplasmic Ca

concentration promotes the binding of Ca

to the calcium-binding

protein calmodulin, which regulates activities of other enzymes, including calcium-dependent

protein kinases.

Figure 2–14.

The Ca

-phosphoinositide signaling pathway. Key proteins include hormone receptors (R), a G

protein (G), a phosphoinositide-specific phospholipase C (PLC), protein kinase C substrates of the

kinase (S), calmodulin (CaM), and calmodulin-binding enzymes (E), including kinases,

phosphodiesterases, etc. (PIP

, phosphatidylinositol-4,5-bisphosphate; DAG, diacylglycerol.

Asterisk denotes activated state. Open arrows denote regulatory effects.)

With its multiple second messengers and protein kinases, the phosphoinositide signaling pathway is

much more complex than the cAMP pathway. For example, different cell types may contain one or

more specialized calcium- and calmodulin-dependent kinases with limited substrate specificity (eg,

myosin light chain kinase) in addition to a general calcium- and calmodulin-dependent kinase that

can phosphorylate a wide variety of protein substrates. Furthermore, at least nine structurally

distinct types of protein kinase C have been identified.

As in the cAMP system, multiple mechanisms damp or terminate signaling by this pathway. IP

inactivated by dephosphorylation; diacylglycerol is either phosphorylated to yield phosphatidic

acid, which is then converted back into phospholipids, or it is deacylated to yield arachidonic acid;

is actively removed from the cytoplasm by Ca

pumps.

These and other nonreceptor elements of the calcium-phosphoinositide signaling pathway are now

becoming targets for drug development. For example, the therapeutic effects of lithium ion, an

established agent for treating manic-depressive illness, may be mediated by effects on the

metabolism of phosphoinositides (see Chapter 29: Antipsychotic Agents & Lithium).

Cyclic Guanosine Monophosphate (cGMP)

Unlike cAMP, the ubiquitous and versatile carrier of diverse messages, cGMP has established

signaling roles in only a few cell types. In intestinal mucosa and vascular smooth muscle, the

cGMP-based signal transduction mechanism closely parallels the cAMP-mediated signaling

mechanism. Ligands detected by cell surface receptors stimulate membrane-bound guanylyl cyclase

to produce cGMP, and cGMP acts by stimulating a cGMP-dependent protein kinase. The actions of

cGMP in these cells are terminated by enzymatic degradation of the cyclic nucleotide and by

dephosphorylation of kinase substrates.

Increased cGMP concentration causes relaxation of vascular smooth muscle by a kinase-mediated

mechanism that results in dephosphorylation of myosin light chains (see Figure 12–2). In these

smooth muscle cells, cGMP synthesis can be elevated by two different transmembrane signaling

mechanisms utilizing two different guanylyl cyclases. ANP, a blood-borne peptide hormone,

stimulates a transmembrane receptor by binding to its extracellular domain, thereby activating the

guanylyl cyclase activity that resides in the receptor's intracellular domain. The other mechanism

mediates responses to NO (see Chapter 19: Nitric Oxide, Donors, & Inhibitors), which is generated

in vascular endothelial cells in response to natural vasodilator agents such as acetylcholine and

histamine (NO is also called endothelium-derived relaxing factor [EDRF]). After entering the target

cell, NO binds to and activates a cytoplasmic guanylyl cyclase. A number of useful vasodilating

drugs act by generating or mimicking NO, or by interfering with the metabolic breakdown of cGMP

by phosphodiesterase (see Chapter 11: Antihypertensive Agents and Chapter 12: Vasodilators & the

Treatment of Angina Pectoris).

Interplay among Signaling Mechanisms

The calcium-phosphoinositide and cAMP signaling pathways oppose one another in some cells and

are complementary in others. For example, vasopressor agents that contract smooth muscle act by

-mediated mobilization of Ca

, whereas agents that relax smooth muscle often act by elevation

of cAMP. In contrast, cAMP and phosphoinositide second messengers act together to stimulate

glucose release from the liver.

Phosphorylation: A Common Theme

Almost all second messenger signaling involves reversible phosphorylation, which performs two

principal functions in signaling: amplification and flexible regulation. In amplification, rather like

GTP bound to a G protein, the attachment of a phosphoryl group to a serine, threonine, or tyrosine

residue powerfully amplifies the initial regulatory signal by recording a molecular memory that the

pathway has been activated; dephosphorylation erases the memory, taking a longer time to do so

than is required for dissociation of an allosteric ligand. In flexible regulation, differing substrate

specificities of the multiple protein kinases regulated by second messengers provide branch points

in signaling pathways that may be independently regulated. In this way, cAMP, Ca

, or other

second messengers can use the presence or absence of particular kinases or kinase substrates to

produce quite different effects in different cell types. Inhibitors of protein kinases have great

potential as therapeutic agents, particularly in neoplastic diseases. Trastuzumab, an antibody that

antagonizes growth factor receptor signaling, was discussed earlier as a therapeutic agent for breast

cancer. Another example of this general approach is imatinib (Gleevec, STI571), a small molecule

inhibitor of the cytoplasmic tyrosine kinase Bcr/Abl, which is activated by growth factor signaling

pathways and is overexpressed in chronic myelogenous leukemia (CML). This compound, a

promising agent for treating CML, was recently approved by the US Food and Drug Administration

(FDA) for clinical use.

Receptor Classes & Drug Development

The existence of a specific drug receptor is usually inferred from studying the structure-activity

relationship of a group of structurally similar congeners of the drug that mimic or antagonize its

effects. Thus, if a series of related agonists exhibits identical relative potencies in producing two

distinct effects, it is likely that the two effects are mediated by similar or identical receptor

molecules. In addition, if identical receptors mediate both effects, a competitive antagonist will

inhibit both responses with the same K

; a second competitive antagonist will inhibit both responses

with its own characteristic K

. Thus, studies of the relation between structure and activity of a series

of agonists and antagonists can identify a species of receptor that mediates a set of pharmacologic

responses.

Exactly the same experimental procedure can show that observed effects of a drug are mediated by

different receptors. In this case, effects mediated by different receptors may exhibit different orders

of potency among agonists and different K

values for each competitive antagonist.

Wherever we look, evolution has created many different receptors that function to mediate

responses to any individual chemical signal. In some cases, the same chemical acts on completely

different structural receptor classes. For example, acetylcholine uses ligand-gated ion channels

(nicotinic AChRs) to initiate a fast excitatory postsynaptic potential (EPSP) in postganglionic

neurons. Acetylcholine also activates a separate class of G protein–coupled receptors (muscarinic

AChRs), which modulate responsiveness of the same neurons to the fast EPSP. In addition, each

structural class usually includes multiple subtypes of receptor, often with significantly different

signaling or regulatory properties. For example, norepinephrine activates many structurally related

receptors, including -adrenergic (stimulation of G

, increased heart rate),

-adrenergic

(stimulation of G

, vasoconstriction), and

-adrenergic (stimulation of G

, opening of K

channels)

(see Table 2–2). The existence of multiple receptor classes and subtypes for the same endogenous

ligand has created important opportunities for drug development. For example, propranolol, a

selective antagonist of -adrenergic receptors, can reduce an accelerated heart rate without

preventing the sympathetic nervous system from causing vasoconstriction, an effect mediated by

receptors.

The principle of drug selectivity may even apply to structurally identical receptors expressed in

different cells, eg, receptors for steroids such as estrogen (Figure 2–6). Different cell types express

different accessory proteins, which interact with steroid receptors and change the functional effects

of drug-receptor interaction. For example, tamoxifen acts as an antagonist on estrogen receptors

expressed in mammary tissue but as an agonist on estrogen receptors in bone. Consequently,

tamoxifen may be useful not only in the treatment and prophylaxis of breast cancer but also in the

prevention of osteoporosis by increasing bone density (see Chapter 40: The Gonadal Hormones &

Inhibitors and Chapter 42: Agents That Affect Bone Mineral Homeostasis). Tamoxifen may also

create complications in postmenopausal women, however, by exerting an agonist action in the

uterus, stimulating endometrial cell proliferation.

New drug development is not confined to agents that act on receptors for extracellular chemical

signals. Pharmaceutical chemists are now determining whether elements of signaling pathways

distal to the receptors may also serve as targets of selective and useful drugs. For example,

clinically useful agents might be developed that act selectively on specific G proteins, kinases,

phosphatases, or the enzymes that degrade second messengers.

Relation between Drug Dose & Clinical Response

We have dealt with receptors as molecules and shown how receptors can quantitatively account for

the relation between dose or concentration of a drug and pharmacologic responses, at least in an

idealized system. When faced with a patient who needs treatment, the prescriber must make a

choice among a variety of possible drugs and devise a dosage regimen that is likely to produce

maximal benefit and minimal toxicity. In order to make rational therapeutic decisions, the

prescriber must understand how drug-receptor interactions underlie the relations between dose and

response in patients, the nature and causes of variation in pharmacologic responsiveness, and the

clinical implications of selectivity of drug action.

Dose & Response in Patients

Graded Dose-Response Relations

To choose among drugs and to determine appropriate doses of a drug, the prescriber must know the

relative pharmacologic potency and maximal efficacy of the drugs in relation to the desired

therapeutic effect. These two important terms, often confusing to students and clinicians, can be

explained by refering to Figure 2–15, which depicts graded dose-response curves that relate dose of

four different drugs to the magnitude of a particular therapeutic effect.

Figure 2–15.

Graded dose-response curves for four drugs, illustrating different pharmacologic potencies and

different maximal efficacies. (See text.)

Potency

Drugs A and B are said to be more potent than drugs C and D because of the relative positions of

their dose-response curves along the dose axis of Figure 2–15. Potency refers to the concentration

(EC

) or dose (ED

) of a drug required to produce 50% of that drug's maximal effect. Thus, the

pharmacologic potency of drug A in Figure 2–15 is less than that of drug B, a partial agonist,

because the EC

of A is greater than the EC

of B. Potency of a drug depends in part on the

affinity (K

) of receptors for binding the drug and in part on the efficiency with which drug-

receptor interaction is coupled to response. Note that some doses of drug A can produce larger

effects than any dose of drug B, despite the fact that we describe drug B as pharmacologically more

potent. The reason for this is that drug A has a larger maximal efficacy, as described below.

For clinical use, it is important to distinguish between a drug's potency and its efficacy. The clinical

effectiveness of a drug depends not on its potency (EC

), but on its maximal efficacy (see below)

and its ability to reach the relevant receptors. This ability can depend on its route of administration,

absorption, distribution through the body, and clearance from the blood or site of action. In deciding

which of two drugs to administer to a patient, the prescriber must usually consider their relative

effectiveness rather than their relative potency. Pharmacologic potency can largely determine the

administered dose of the chosen drug.

For therapeutic purposes, the potency of a drug should be stated in dosage units, usually in terms of

a particular therapeutic end point (eg, 50 mg for mild sedation, 1

g/kg/min for an increase in heart

rate of 25 beats/min). Relative potency, the ratio of equi-effective doses (0.2, 10, etc), may be used

in comparing one drug with another.

Maximal Efficacy

This parameter reflects the limit of the dose-response relation on the response axis. Drugs A, C,

and D in Figure 2–15 have equal maximal efficacy, while all have greater maximal efficacy than

drug B. The maximal efficacy (sometimes referred to simply as efficacy) of a drug is obviously

crucial for making clinical decisions when a large response is needed. It may be determined by the

drug's mode of interactions with receptors (as with partial agonists, described above)

or by

characteristics of the receptor-effector system involved.

Note that "maximal efficacy," used in a therapeutic context, does not have exactly the same

meaning the term denotes in the more specialized context of drug-receptor interactions described

earlier in this chapter. In an idealized in vitro system, efficacy denotes the relative maximal efficacy

of agonists and partial agonists that act via the same receptor. In therapeutics, efficacy denotes the

extent or degree of an effect that can be achieved in the intact patient. Thus, therapeutic efficacy

may be affected by the characteristics of a particular drug-receptor interaction, but it also depends

on a host of other factors as noted in the text.

Thus, diuretics that act on one portion of the nephron may produce much greater excretion of fluid

and electrolytes than diuretics that act elsewhere. In addition, the practical efficacy of a drug for

achieving a therapeutic end point (eg, increased cardiac contractility) may be limited by the drug's

propensity to cause a toxic effect (eg, fatal cardiac arrhythmia) even if the drug could otherwise

produce a greater therapeutic effect.

Shape of Dose-Response Curves

While the responses depicted in curves A, B, and C of Figure 2–15 approximate the shape of a

simple Michaelis-Menten relation (transformed to a logarithmic plot), some clinical responses do

not. Extremely steep dose-response curves (eg, curve D) may have important clinical consequences

if the upper portion of the curve represents an undesirable extent of response (eg, coma caused by a

sedative-hypnotic). Steep dose-response curves in patients could result from cooperative

interactions of several different actions of a drug (eg, effects on brain, heart, and peripheral vessels,

all contributing to lowering of blood pressure).

Quantal Dose-Effect Curves

Graded dose-response curves of the sort described above have certain limitations in their

application to clinical decision making. For example, such curves may be impossible to construct if

the pharmacologic response is an either-or (quantal) event, such as prevention of convulsions,

arrhythmia, or death. Furthermore, the clinical relevance of a quantitative dose-response

relationship in a single patient, no matter how precisely defined, may be limited in application to

other patients, owing to the great potential variability among patients in severity of disease and

responsiveness to drugs.

Some of these difficulties may be avoided by determining the dose of drug required to produce a

specified magnitude of effect in a large number of individual patients or experimental animals and

plotting the cumulative frequency distribution of responders versus the log dose (Figure 2–16). The

specified quantal effect may be chosen on the basis of clinical relevance (eg, relief of headache) or

for preservation of safety of experimental subjects (eg, using low doses of a cardiac stimulant and

specifying an increase in heart rate of 20 beats/min as the quantal effect), or it may be an inherently

quantal event (eg, death of an experimental animal). For most drugs, the doses required to produce a

specified quantal effect in individuals are lognormally distributed; ie, a frequency distribution of

such responses plotted against the log of the dose produces a gaussian normal curve of variation

(colored area, Figure 2–16). When these responses are summated, the resulting cumulative

frequency distribution constitutes a quantal dose-effect curve (or dose-percent curve) of the

proportion or percentage of individuals who exhibit the effect plotted as a function of log dose

(Figure 2–16).

Figure 2–16.

Quantal dose-effect plots. Shaded boxes (and the accompanying curves) indicate the frequency

distribution of doses of drug required to produce a specified effect; ie, the percentage of animals

that required a particular dose to exhibit the effect. The open boxes (and the corresponding curves)

indicate the cumulative frequency distribution of responses, which are lognormally distributed.

The quantal dose-effect curve is often characterized by stating the median effective dose (ED

the dose at which 50% of individuals exhibit the specified quantal effect. (Note that the abbreviation

has a different meaning in this context from its meaning in relation to graded dose-effect

curves, described above.) Similarly, the dose required to produce a particular toxic effect in 50% of

animals is called the median toxic dose (TD

) If the toxic effect is death of the animal, a median

lethal dose (LD

) may be experimentally defined. Such values provide a convenient way of

comparing the potencies of drugs in experimental and clinical settings: Thus, if the ED

s of two

drugs for producing a specified quantal effect are 5 and 500 mg, respectively, then the first drug can

be said to be 100 times more potent than the second for that particular effect. Similarly, one can

obtain a valuable index of the selectivity of a drug's action by comparing its ED

s for two different

quantal effects in a population (eg, cough suppression versus sedation for opioid drugs).

Quantal dose-effect curves may also be used to generate information regarding the margin of safety

to be expected from a particular drug used to produce a specified effect. One measure, which relates

the dose of a drug required to produce a desired effect to that which produces an undesired effect, is

the therapeutic index. In animal studies, the therapeutic index is usually defined as the ratio of the

to the ED

for some therapeutically relevant effect. The precision possible in animal

experiments may make it useful to use such a therapeutic index to estimate the potential benefit of a

drug in humans. Of course, the therapeutic index of a drug in humans is almost never known with

real precision; instead, drug trials and accumulated clinical experience often reveal a range of

usually effective doses and a different (but sometimes overlapping) range of possibly toxic doses.

The clinically acceptable risk of toxicity depends critically on the severity of the disease being

treated. For example, the dose range that provides relief from an ordinary headache in the great

majority of patients should be very much lower than the dose range that produces serious toxicity,

even if the toxicity occurs in a small minority of patients. However, for treatment of a lethal disease

such as Hodgkin's lymphoma, the acceptable difference between therapeutic and toxic doses may be

smaller.

Finally, note that the quantal dose-effect curve and the graded dose-response curve summarize

somewhat different sets of information, although both appear sigmoid in shape on a

semilogarithmic plot (compare Figures 2–15 and 2–16). Critical information required for making

rational therapeutic decisions can be obtained from each type of curve. Both curves provide

information regarding the potency and selectivity of drugs; the graded dose-response curve

indicates the maximal efficacy of a drug, and the quantal dose-effect curve indicates the potential

variability of responsiveness among individuals.

Variation in Drug Responsiveness

Individuals may vary considerably in their responsiveness to a drug; indeed, a single individual may

respond differently to the same drug at different times during the course of treatment. Occasionally,

individuals exhibit an unusual or idiosyncratic drug response, one that is infrequently observed in

most patients. The idiosyncratic responses are usually caused by genetic differences in metabolism

of the drug or by immunologic mechanisms, including allergic reactions.

Quantitative variations in drug response are in general more common and more clinically important.

An individual patient is hyporeactive or hyperreactive to a drug in that the intensity of effect of a

given dose of drug is diminished or increased in comparison to the effect seen in most individuals.

(Note: The term hypersensitivity usually refers to allergic or other immunologic responses to

drugs.) With some drugs, the intensity of response to a given dose may change during the course of

therapy; in these cases, responsiveness usually decreases as a consequence of continued drug

administration, producing a state of relative tolerance to the drug's effects. When responsiveness

diminishes rapidly after administration of a drug, the response is said to be subject to

tachyphylaxis.

Even before administering the first dose of a drug, the prescriber should consider factors that may

help in predicting the direction and extent of possible variations in responsiveness. These include

the propensity of a particular drug to produce tolerance or tachyphylaxis as well as the effects of

age, sex, body size, disease state, genetic factors, and simultaneous administration of other drugs.

Four general mechanisms may contribute to variation in drug responsiveness among patients or

within an individual patient at different times.

Alteration in Concentration of Drug That Reaches the Receptor

Patients may differ in the rate of absorption of a drug, in distributing it through body compartments,

or in clearing the drug from the blood (see Chapter 3: Pharmacokinetics & Pharmacodynamics:

Rational Dosing & the Time Course of Drug Action). By altering the concentration of drug that

reaches relevant receptors, such pharmacokinetic differences may alter the clinical response. Some

differences can be predicted on the basis of age, weight, sex, disease state, liver and kidney

function, and by testing specifically for genetic differences that may result from inheritance of a

functionally distinctive complement of drug-metabolizing enzymes (see Chapter 3:

Pharmacokinetics & Pharmacodynamics: Rational Dosing & the Time Course of Drug Action and

Chapter 4: Drug Biotransformation).

Variation in Concentration of an Endogenous Receptor Ligand

This mechanism contributes greatly to variability in responses to pharmacologic antagonists. Thus,

propranolol, a -adrenoceptor antagonist, will markedly slow the heart rate of a patient whose

endogenous catecholamines are elevated (as in pheochromocytoma) but will not affect the resting

heart rate of a well-trained marathon runner. A partial agonist may exhibit even more dramatically

different responses: Saralasin, a weak partial agonist at angiotensin II receptors, lowers blood

pressure in patients with hypertension caused by increased angiotensin II production and raises

blood pressure in patients who produce small amounts of angiotensin.

Alterations in Number or Function of Receptors

Experimental studies have documented changes in drug responsiveness caused by increases or

decreases in the number of receptor sites or by alterations in the efficiency of coupling of receptors

to distal effector mechanisms. In some cases, the change in receptor number is caused by other

hormones; for example, thyroid hormones increase both the number of receptors in rat heart

muscle and cardiac sensitivity to catecholamines. Similar changes probably contribute to the

tachycardia of thyrotoxicosis in patients and may account for the usefulness of propranolol, a -

adrenoceptor antagonist, in ameliorating symptoms of this disease.

In other cases, the agonist ligand itself induces a decrease in the number (eg, down-regulation) or

coupling efficiency (eg, desensitization) of its receptors. These mechanisms (discussed above, under

Signaling Mechanisms & Drug Actions) may contribute to two clinically important phenomena:

first, tachyphylaxis or tolerance to the effects of some drugs (eg, biogenic amines and their

congeners), and second, the "overshoot" phenomena that follow withdrawal of certain drugs. These

phenomena can occur with either agonists or antagonists. An antagonist may increase the number of

receptors in a critical cell or tissue by preventing down-regulation caused by an endogenous

agonist. When the antagonist is withdrawn, the elevated number of receptors can produce an

exaggerated response to physiologic concentrations of agonist. Potentially disastrous withdrawal

symptoms can result for the opposite reason when administration of an agonist drug is discontinued.

In this situation, the number of receptors, which has been decreased by drug-induced down-

regulation, is too low for endogenous agonist to produce effective stimulation. For example, the

withdrawal of clonidine (a drug whose

-adrenoceptor agonist activity reduces blood pressure) can

produce hypertensive crisis, probably because the drug down-regulates

-adrenoceptors (see

Chapter 11: Antihypertensive Agents).

Genetic factors also can play an important role in altering the number or function of specific

receptors. For example, a specific genetic variant of the

-adrenoceptor—when inherited together

with a specific variant of the

-adrenoceptor—confers a greatly increased risk for developing

congestive heart failure which may be reduced by early intervention using antagonist drugs. The

identification of such genetic factors, part of the rapidly developing field of pharmacogenetics,

holds exciting promise for clinical diagnosis and may help physicians design the most appropriate

pharmacologic therapy for individual patients.

Changes in Components of Response Distal to the Receptor

Although a drug initiates its actions by binding to receptors, the response observed in a patient

depends on the functional integrity of biochemical processes in the responding cell and physiologic

regulation by interacting organ systems. Clinically, changes in these postreceptor processes

represent the largest and most important class of mechanisms that cause variation in responsiveness

to drug therapy.

Before initiating therapy with a drug, the prescriber should be aware of patient characteristics that

may limit the clinical response. These characteristics include the age and general health of the

patient and—most importantly—the severity and pathophysiologic mechanism of the disease. The

most important potential cause of failure to achieve a satisfactory response is that the diagnosis is

wrong or physiologically incomplete. Drug therapy will always be most successful when it is

accurately directed at the pathophysiologic mechanism responsible for the disease.

When the diagnosis is correct and the drug is appropriate, an unsatisfactory therapeutic response

can often be traced to compensatory mechanisms in the patient that respond to and oppose the

beneficial effects of the drug. Compensatory increases in sympathetic nervous tone and fluid

retention by the kidney, for example, can contribute to tolerance to antihypertensive effects of a

vasodilator drug. In such cases, additional drugs may be required to achieve a useful therapeutic

response.

Clinical Selectivity: Beneficial Versus Toxic Effects of Drugs

Although we classify drugs according to their principal actions, it is clear that no drug causes only a

single, specific effect. Why is this so? It is exceedingly unlikely that any kind of drug molecule will

bind to only a single type of receptor molecule, if only because the number of potential receptors in

every patient is astronomically large. Even if the chemical structure of a drug allowed it to bind to

only one kind of receptor, the biochemical processes controlled by such receptors would take place

in multiple cell types and would be coupled to many other biochemical functions; as a result, the

patient and the prescriber would probably perceive more than one drug effect. Accordingly, drugs

are only selective—rather than specific—in their actions, because they bind to one or a few types of

receptor more tightly than to others and because these receptors control discrete processes that

result in distinct effects.

It is only because of their selectivity that drugs are useful in clinical medicine. Selectivity can be

measured by comparing binding affinities of a drug to different receptors or by comparing ED

s for

different effects of a drug in vivo. In drug development and in clinical medicine, selectivity is

usually considered by separating effects into two categories: beneficial or therapeutic effects

versus toxic effects. Pharmaceutical advertisements and prescribers occasionally use the term side

effect, implying that the effect in question is insignificant or occurs via a pathway that is to one side

of the principal action of the drug; such implications are frequently erroneous.

Beneficial and Toxic Effects Mediated by the Same Receptor-Effector Mechanism

Much of the serious drug toxicity in clinical practice represents a direct pharmacologic extension of

the therapeutic actions of the drug. In some of these cases (bleeding caused by anticoagulant

therapy; hypoglycemic coma due to insulin), toxicity may be avoided by judicious management of

the dose of drug administered, guided by careful monitoring of effect (measurements of blood

coagulation or serum glucose) and aided by ancillary measures (avoiding tissue trauma that may

lead to hemorrhage; regulation of carbohydrate intake). In still other cases, the toxicity may be

avoided by not administering the drug at all, if the therapeutic indication is weak or if other therapy

is available.

In certain situations, a drug is clearly necessary and beneficial but produces unacceptable toxicity

when given in doses that produce optimal benefit. In such situations, it may be necessary to add

another drug to the treatment regimen. In treating hypertension, for example, administration of a

second drug often allows the prescriber to reduce the dose and toxicity of the first drug (see Chapter

11: Antihypertensive Agents).

Beneficial and Toxic Effects Mediated by Identical Receptors But in Different Tissues or by

Different Effector Pathways

Many drugs produce both their desired effects and adverse effects by acting on a single receptor

type in different tissues. Examples discussed in this book include: digitalis glycosides, which act by

inhibiting Na

ATPase in cell membranes; methotrexate, which inhibits the enzyme

dihydrofolate reductase; and glucocorticoid hormones.

Three therapeutic strategies are used to avoid or mitigate this sort of toxicity. First, the drug should

always be administered at the lowest dose that produces acceptable benefit. Second, adjunctive

drugs that act through different receptor mechanisms and produce different toxicities may allow

lowering the dose of the first drug, thus limiting its toxicity (eg, use of other immunosuppressive

agents added to glucocorticoids in treating inflammatory disorders). Third, selectivity of the drug's

actions may be increased by manipulating the concentrations of drug available to receptors in

different parts of the body, for example, by aerosol administration of a glucocorticoid to the bronchi

in asthma.

Beneficial and Toxic Effects Mediated by Different Types of Receptors

Therapeutic advantages resulting from new chemical entities with improved receptor selectivity

were mentioned earlier in this chapter and are described in detail in later chapters. Such drugs

include the

- and -selective adrenoceptor agonists and antagonists, the H

and H

antihistamines,

nicotinic and muscarinic blocking agents, and receptor-selective steroid hormones. All of these

receptors are grouped in functional families, each responsive to a small class of endogenous

agonists. The receptors and their associated therapeutic uses were discovered by analyzing effects

of the physiologic chemical signals—catecholamines, histamine, acetylcholine, and corticosteroids.

A number of other drugs were discovered by exploiting therapeutic or toxic effects of chemically

similar agents observed in a clinical context. Examples include quinidine, the sulfonylureas,

thiazide diuretics, tricyclic antidepressants, opioid drugs, and phenothiazine antipsychotics. Often

the new agents turn out to interact with receptors for endogenous substances (eg, opioids and

phenothiazines for endogenous opioid and dopamine receptors, respectively). It is likely that other

new drugs will be found to do so in the future, perhaps leading to the discovery of new classes of

receptors and endogenous ligands for future drug development.

Thus, the propensity of drugs to bind to different classes of receptor sites is not only a potentially

vexing problem in treating patients, it also presents a continuing challenge to pharmacology and an

opportunity for developing new and more useful drugs.

Chapter 3. Pharmacokinetics & Pharmacodynamics: Rational

Dosing & the Time Course of Drug Action

Pharmacokinetics & Pharmacodynamics: Rational Dosing & the Time Course of Drug Action:

Introduction

The goal of therapeutics is to achieve a desired beneficial effect with minimal adverse effects.

When a medicine has been selected for a patient, the clinician must determine the dose that most

closely achieves this goal. A rational approach to this objective combines the principles of

pharmacokinetics with pharmacodynamics to clarify the dose-effect relationship (Figure 3–1).

Pharmacodynamics governs the concentration-effect part of the interaction, whereas

pharmacokinetics deals with the dose-concentration part (Holford & Sheiner, 1981). The

pharmacokinetic processes of absorption, distribution, and elimination determine how rapidly and

for how long the drug will appear at the target organ. The pharmacodynamic concepts of maximum

response and sensitivity determine the magnitude of the effect at a particular concentration (see E

max

and EC

, Chapter 2: Drug Receptors & Pharmacodynamics).

Figure 3–1.

The relationship between dose and effect can be separated into pharmacokinetic (dose-

concentration) and pharmacodynamic (concentration-effect) components. Concentration provides

the link between pharmacokinetics and pharmacodynamics and is the focus of the target

concentration approach to rational dosing. The three primary processes of pharmacokinetics are

absorption, distribution, and elimination.

Figure 3–1 illustrates a fundamental hypothesis of pharmacology, namely, that a relationship exists

between a beneficial or toxic effect of a drug and the concentration of the drug. This hypothesis has

been documented for many drugs, as indicated by the Target Concentrations and Toxic

Concentrations columns in Table 3–1. The apparent lack of such a relationship for some drugs does

not weaken the basic hypothesis but points to the need to consider the time course of concentration

at the actual site of pharmacologic effect (see below).

Table 3–1. Pharmacokinetic and Pharmacodynamic Parameters for Selected Drugs. (See Speight &

Holford, 1997, for a More Comprehensive Listing.)

Drug Oral

Availabi

lity (F)

(%)

Urinar

Excreti

on (%)

Boun

d in

Plas

(%)

Cleara

nce

(L/h/70

kg)

Volume

Distribut

ion (L/70

kg)

Half-

Life (h)

Target

Concentrat

ions

Toxic

Concentrat

ions

Acetaminoph

88 3 0 21 67 2 15 mg/L >300 mg/L

Acyclovir 23 75 15 19.8 48 2.4 . . . . . .

Amikacin . . . 98 4 5.46 19 2.3 . . . . . .

Amoxicillin 93 86 18 10.8 15 1.7 . . . . . .

Amphoterici

. . . 4 90 1.92 53 18 . . . . . .

Ampicillin 62 82 18 16.2 20 1.3 . . . . . .

Aspirin 68 1 49 39 11 0.25 . . . . . .

Atenolol 56 94 5 10.2 67 6.1 1 mg/L . . .

Atropine 50 57 18 24.6 120 4.3 . . . . . .

Captopril 65 38 30 50.4 57 2.2 50 ng/mL . . .

Carbamazepi

70 1 74 5.34 98 15 6 mg/L >9 mg/L

Cephalexin 90 91 14 18 18 0.9 . . . . . .

Cephalothin . . . 52 71 28.2 18 0.57 . . . . . .

Chloramphen

icol

80 25 53 10.2 66 2.7 . . . . . .

Chlordiazepo

xide

100 1 97 2.28 21 10 1 mg/L . . .

Chloroquine 89 61 61 45 13000 214 20 ng/mL 250 ng/mL

Chlorpropam

ide

90 20 96 0.126 6.8 33 . . . . . .

Cimetidine 62 62 19 32.4 70 1.9 0.8 mg/L . . .

Ciprofloxaci

60 65 40 25.2 130 4.1 . . . . . .

Clonidine 95 62 20 12.6 150 12 1 ng/mL . . .

Cyclosporine 23 1 93 24.6 85 5.6 200 ng/mL >400 ng/mL

Diazepam 100 1 99 1.62 77 43 300 ng/mL . . .

Digitoxin 90 32 97 0.234 38 161 10 ng/mL >35 ng/mL

Digoxin 70 60 25 7 500 50 1 ng/mL >2 ng/mL

Diltiazem 44 4 78 50.4 220 3.7 . . . . . .

Disopyramid

83 55

5.04 41 6 3 mg/L >8 mg/L

Enalapril 95 90 55 9 40 3 > 0.5 ng/mL . . .

Erythromyci

35 12 84 38.4 55 1.6 . . . . . .

Ethambutol 77 79 5 36 110 3.1 . . . >10 mg/L

Fluoxetine 60 3 94 40.2 2500 53 . . . . . .

Furosemide 61 66 99 8.4 7.7 1.5 . . . >25 mg/L

Gentamicin . . . 90 10 5.4 18 2.5 . . . . . .

Hydralazine 40 10 87 234 105 1 100 ng/mL . . .

Imipramine 40 2 90 63 1600 18 200 ng/mL >1 mg/L

Indomethacin 98 15 90 8.4 18 2.4 1 mg/L >5 mg/L

Labetalol 18 5 50 105 660 4.9 0.1 mg/L . . .

Lidocaine 35 2 70 38.4 77 1.8 3 mg/L >6 mg/L

Lithium 100 95 0 1.5 55 22 0.7 mEq/L >2 mEq/L

Meperidine 52 12 58 72 310 3.2 0.5 mg/L . . .

Methotrexate 70 48 34 9 39 7.2 750 M-h

>950 M-h

Metoprolol 38 10 11 63 290 3.2 25 ng/mL . . .

Metronidazol

99 10 10 5.4 52 8.5 4 mg/L . . .

Midazolam 44 56 95 27.6 77 1.9 . . . . . .

Morphine 24 8 35 60 230 1.9 60 ng/mL . . .

Nifedipine 50 0 96 29.4 55 1.8 50 ng/mL . . .

Nortriptyline 51 2 92 30 1300 31 100 ng/mL >500 ng/mL

Phenobarbita

100 24 51 0.258 38 98 15 mg/L >30 mg/L

Phenytoin 90 2 89 Conc-

depende

45 Conc-

depende

10 mg/L >20 mg/L

Prazosin 68 1 95 12.6 42 2.9 . . . . . .

Procainamide 83 67 16 36 130 3 5 mg/L >14 mg/L

Propranolol 26 1 87 50.4 270 3.9 20 ng/mL . . .

Pyridostigmi

14 85 . . . 36 77 1.9 75 ng/mL . . .

Quinidine 80 18 87 19.8 190 6.2 3 mg/L >8 mg/L

Ranitidine 52 69 15 43.8 91 2.1 100 ng/mL . . .

Rifampin ? 7 89 14.4 68 3.5 . . . . . .

Salicylic acid 100 15 85 0.84 12 13 200 mg/L >200 mg/L

Sulfamethox

azole

100 14 62 1.32 15 10 . . . . . .

Terbutaline 14 56 20 14.4 125 14 2 ng/mL . . .

Tetracycline 77 58 65 7.2 105 11 . . . . . .

Theophylline 96 18 56 2.8 35 8.1 10 mg/L >20 mg/L

Tobramycin . . . 90 10 4.62 18 2.2 . . . . . .

Tocainide 89 38 10 10.8 210 14 10 mg/L . . .

Tolbutamide 93 0 96 1.02 7 5.9 100 mg/L . . .

Trimethopri

100 69 44 9 130 11 . . . . . .

Tubocurarine . . . 63 50 8.1 27 2 0.6 mg/L . . .

Valproic acid 100 2 93 0.462 9.1 14 75 mg/L >150 mg/L

Vancomycin . . . 79 30 5.88 27 5.6 . . . . . .

Verapamil 22 3 90 63 350 4 . . . . . .

Warfarin 93 3 99 0.192 9.8 37 . . . . . .

Zidovudine 63 18 25 61.8 98 1.1 . . . . . .

Convert to mL/min by multiplying the number given by 16.6.

Varies with concentration.

Target area under the concentration time curve after a single dose.

Can be estimated from measured C

using CL = V

max

/(K

+ C

); V

max

= 415 mg/d, K

= 5 mg/L.

See text.

Varies because of concentration-dependent clearance.

Knowing the relationship between dose, drug concentration and effects allows the clinician to take

into account the various pathologic and physiologic features of a particular patient that make him or

her different from the average individual in responding to a drug. The importance of

pharmacokinetics and pharmacodynamics in patient care thus rests upon the improvement in

therapeutic benefit and reduction in toxicity that can be achieved by application of these principles.

Pharmacokinetics

The "standard" dose of a drug is based on trials in healthy volunteers and patients with average

ability to absorb, distribute, and eliminate the drug (see Clinical Trials: The IND and NDA in

Chapter 5: Basic & Clinical Evaluation of New Drugs). This dose will not be suitable for every

patient. Several physiologic processes (eg, maturation of organ function in infants) and pathologic

processes (eg, heart failure, renal failure) dictate dosage adjustment in individual patients. These

processes modify specific pharmacokinetic parameters. The two basic parameters are clearance, the

measure of the ability of the body to eliminate the drug; and volume of distribution, the measure of

the apparent space in the body available to contain the drug. These parameters are illustrated

schematically in Figure 3–2, where the volume of the compartments into which the drugs diffuse

represents the volume of distribution and the size of the outflow "drain" in Figures 3–2 B and D

represents the clearance.

Figure 3–2.

Models of drug distribution and elimination. The effect of adding drug to the blood by rapid

intravenous injection is represented by expelling a known amount of the agent into a beaker. The

time course of the amount of drug in the beaker is shown in the graphs at the right. In the first

example (A), there is no movement of drug out of the beaker, so the graph shows only a steep rise

to maximum followed by a plateau. In the second example (B), a route of elimination is present,

and the graph shows a slow decay after a sharp rise to a maximum. Because the level of material in

the beaker falls, the "pressure" driving the elimination process also falls, and the slope of the curve

decreases. This is an exponential decay curve. In the third model (C), drug placed in the first

compartment ("blood") equilibrates rapidly with the second compartment ("extravascular volume")

and the amount of drug in "blood" declines exponentially to a new steady state. The fourth model

(D) illustrates a more realistic combination of elimination mechanism and extravascular

equilibration. The resulting graph shows an early distribution phase followed by the slower

elimination phase.

Volume of Distribution

Volume of distribution (V

) relates the amount of drug in the body to the concentration of drug (C)

in blood or plasma:

The volume of distribution may be defined with respect to blood, plasma, or water (unbound drug),

depending on the concentration used in equation (1) (C = C

, C

, or C

That the V

calculated from equation (1) is an apparent volume may be appreciated by comparing

the volumes of distribution of drugs such as digoxin or chloroquine (Table 3–1) with some of the

physical volumes of the body (Table 3–2). Volume of distribution can vastly exceed any physical

volume in the body because it is the volume apparently necessary to contain the amount of drug

homogeneously at the concentration found in the blood, plasma, or water. Drugs with very high

volumes of distribution have much higher concentrations in extravascular tissue than in the vascular

compartment, ie, they are not homogeneously distributed. Drugs that are completely retained within

the vascular compartment, on the other hand, have a minimum possible volume of distribution equal

to the blood component in which they are distributed, eg, 0.04 L/kg body weight or 2.8 L/70 kg

(Table 3–2) for a drug that is restricted to the plasma compartment.

Table 3–2. Physical Volumes (in L/Kg Body Weight) of Some Body Compartments into Which

Drugs May Be Distributed.

Compartment and Volume Examples of Drugs

Water

Total body water (0.6 L/kg

)

Small water-soluble molecules: eg, ethanol.

Extracellular water (0.2 L/kg) Larger water-soluble molecules: eg, gentamicin.

Blood (0.08 L/kg); plasma (0.04

L/kg)

Strongly plasma protein-bound molecules and very large

molecules: eg, heparin.

Fat (0.2–0.35 L/kg) Highly lipid-soluble molecules: eg, DDT.

Bone (0.07 L/kg) Certain ions: eg, lead, fluoride.

An average figure. Total body water in a young lean man might be 0.7 L/kg; in an obese woman,

0.5 L/kg.

Clearance

Drug clearance principles are similar to the clearance concepts of renal physiology. Clearance of a

drug is the factor that predicts the rate of elimination in relation to the drug concentration:

Clearance, like volume of distribution, may be defined with respect to blood (CL

), plasma (CL

or unbound in water (CL

), depending on the concentration measured.

It is important to note the additive character of clearance. Elimination of drug from the body may

involve processes occurring in the kidney, the lung, the liver, and other organs. Dividing the rate of

elimination at each organ by the concentration of drug presented to it yields the respective clearance

at that organ. Added together, these separate clearances equal total systemic clearance:

"Other" tissues of elimination could include the lungs and additional sites of metabolism, eg, blood

or muscle.

The two major sites of drug elimination are the kidneys and the liver. Clearance of unchanged drug

in the urine represents renal clearance. Within the liver, drug elimination occurs via

biotransformation of parent drug to one or more metabolites, or excretion of unchanged drug into

the bile, or both. The pathways of biotransformation are discussed in Chapter 4: Drug

Biotransformation. For most drugs, clearance is constant over the concentration range encountered

in clinical settings, ie, elimination is not saturable, and the rate of drug elimination is directly

proportional to concentration (rearranging equation [2]):

This is usually referred to as first-order elimination. When clearance is first-order, it can be

estimated by calculating the area under the curve (AUC) of the time-concentration profile after a

dose. Clearance is calculated from the dose divided by the AUC.

Capacity-Limited Elimination

For drugs that exhibit capacity-limited elimination (eg, phenytoin, ethanol), clearance will vary

depending on the concentration of drug that is achieved (Table 3–1). Capacity-limited elimination is

also known as saturable, dose- or concentration-dependent, nonlinear, and Michaelis-Menten

elimination.

Most drug elimination pathways will become saturated if the dose is high enough. When blood flow

to an organ does not limit elimination (see below), the relation between elimination rate and

concentration (C) is expressed mathematically in equation (5):

The maximum elimination capacity is V

max

, and K

is the drug concentration at which the rate of

elimination is 50% of V

max

. At concentrations that are high relative to the K

, the elimination rate is

almost independent of concentration—a state of "pseudo-zero order" elimination. If dosing rate

exceeds elimination capacity, steady state cannot be achieved: The concentration will keep on rising

as long as dosing continues. This pattern of capacity-limited elimination is important for three drugs

in common use: ethanol, phenytoin, and aspirin. Clearance has no real meaning for drugs with

capacity-limited elimination, and AUC cannot be used to describe the elimination of such drugs.

Flow-Dependent Elimination

In contrast to capacity-limited drug elimination, some drugs are cleared very readily by the organ of

elimination, so that at any clinically realistic concentration of the drug, most of the drug in the

blood perfusing the organ is eliminated on the first pass of the drug through it. The elimination of

these drugs will thus depend primarily on the rate of drug delivery to the organ of elimination. Such

drugs (see Table 4–7) can be called "high-extraction" drugs since they are almost completely

extracted from the blood by the organ. Blood flow to the organ is the main determinant of drug

delivery, but plasma protein binding and blood cell partitioning may also be important for

extensively bound drugs that are highly extracted.

Half-Life

Half-life (t

1/2

) is the time required to change the amount of drug in the body by one-half during

elimination (or during a constant infusion). In the simplest case—and the most useful in designing

drug dosage regimens—the body may be considered as a single compartment (as illustrated in

Figure 3–2 B) of a size equal to the volume of distribution (V

). The time course of drug in the body

will depend on both the volume of distribution and the clearance:

The constant 0.7 in equation (6) is an approximation to the natural logarithm of 2. Because drug

elimination can be described by an exponential process, the time taken for a twofold decrease can

be shown to be proportional to ln(2).

Half-life is useful because it indicates the time required to attain 50% of steady state—or to decay

50% from steady-state conditions—after a change in the rate of drug administration. Figure 3–3

shows the time course of drug accumulation during a constant-rate drug infusion and the time

course of drug elimination after stopping an infusion that has reached steady state.

Figure 3–3.

The time course of drug accumulation and elimination. Solid line: Plasma concentrations

reflecting drug accumulation during a constant rate infusion of a drug. Fifty percent of the steady-

state concentration is reached after one half-life, 75% after two half-lives, and over 90% after four

half-lives. Dashed line: Plasma concentrations reflecting drug elimination after a constant rate

infusion of a drug had reached steady state. Fifty percent of the drug is lost after one half-life, 75%

after two half-lives, etc. The "rule of thumb" that four half-lives must elapse after starting a drug-

dosing regimen before full effects will be seen is based on the approach of the accumulation curve

to over 90% of the final steady-state concentration.

Disease states can affect both of the physiologically related primary pharmacokinetic parameters:

volume of distribution and clearance. A change in half-life will not necessarily reflect a change in

drug elimination. For example, patients with chronic renal failure have decreased renal clearance of

digoxin but also a decreased volume of distribution; the increase in digoxin half-life is not as great

as might be expected based on the change in renal function. The decrease in volume of distribution

is due to the decreased renal and skeletal muscle mass and consequent decreased tissue binding of

digoxin to Na

ATPase.

Many drugs will exhibit multicompartment pharmacokinetics (as illustrated in Figures 3–2 C and

D). Under these conditions, the "true" terminal half-life, as given in Table 3–1, will be greater than

that calculated from equation (6).

Drug Accumulation

Whenever drug doses are repeated, the drug will accumulate in the body until dosing stops. This is

because it takes an infinite time (in theory) to eliminate all of a given dose. In practical terms, this

means that if the dosing interval is shorter than four half-lives, accumulation will be detectable.

Accumulation is inversely proportional to the fraction of the dose lost in each dosing interval. The

fraction lost is 1 minus the fraction remaining just before the next dose. The fraction remaining can

be predicted from the dosing interval and the half-life. A convenient index of accumulation is the

accumulation factor.

For a drug given once every half-life, the accumulation factor is 1/0.5, or 2. The accumulation

factor predicts the ratio of the steady-state concentration to that seen at the same time following the

first dose. Thus, the peak concentrations after intermittent doses at steady state will be equal to the

peak concentration after the first dose multiplied by the accumulation factor.

Bioavailability

Bioavailability is defined as the fraction of unchanged drug reaching the systemic circulation

following administration by any route (Table 3–3). The area under the blood concentration-time

curve (area under the curve, AUC) is a common measure of the extent of bioavailability for a drug

given by a particular route (Figure 3–4). For an intravenous dose of the drug, bioavailability is

assumed to be equal to unity. For a drug administered orally, bioavailability may be less than 100%

for two main reasons—incomplete extent of absorption and first-pass elimination.

Table 3–3. Routes of Administration, Bioavailability, and General Characteristics.

Route Bioavailability

(%)

Characteristics

Intravenous (IV) 100 (by

definition)

Most rapid onset

Intramuscular

(IM)

75 to 100 Large volumes often feasible; may be painful

Subcutaneous

(SC)

75 to 100 Smaller volumes than IM; may be painful

Oral (PO) 5 to <100 Most convenient; first-pass effect may be significant

Rectal (PR) 30 to <100 Less first-pass effect than oral

Inhalation 5 to <100 Often very rapid onset

Transdermal 80 to 100 Usually very slow absorption; used for lack of first-pass

effect; prolonged duration of action

Figure 3–4.

Blood concentration-time curves, illustrating how changes in the rate of absorption and extent of

bioavailability can influence both the duration of action and the effectiveness of the same total

dose of a drug administered in three different formulations. The dashed line indicates the target

concentration (TC) of the drug in the blood.

Extent of Absorption

After oral administration, a drug may be incompletely absorbed, eg, only 70% of a dose of digoxin

reaches the systemic circulation. This is mainly due to lack of absorption from the gut. Other drugs

are either too hydrophilic (eg, atenolol) or too lipophilic (eg, acyclovir) to be absorbed easily, and

their low bioavailability is also due to incomplete absorption. If too hydrophilic, the drug cannot

cross the lipid cell membrane; if too lipophilic, the drug is not soluble enough to cross the water

layer adjacent to the cell. Drugs may not be absorbed because of a reverse transporter associated

with P-glycoprotein. This process actively pumps drug out of gut wall cells back into the gut lumen.

Inhibition of P-glycoprotein and gut wall metabolism, eg, by grapefruit juice, may be associated

with substantially increased drug absorption.

First-Pass Elimination

Following absorption across the gut wall, the portal blood delivers the drug to the liver prior to

entry into the systemic circulation. A drug can be metabolized in the gut wall (eg, by the CYP3A4

enzyme system) or even in the portal blood, but most commonly it is the liver that is responsible for

metabolism before the drug reaches the systemic circulation. In addition, the liver can excrete the

drug into the bile. Any of these sites can contribute to this reduction in bioavailability, and the

overall process is known as first-pass elimination. The effect of first-pass hepatic elimination on

bioavailability is expressed as the extraction ratio (ER):

where Q is hepatic blood flow, normally about 90 L/h in a person weighing 70 kg.

The systemic bioavailability of the drug (F) can be predicted from the extent of absorption (f) and

the extraction ratio (ER):

A drug such as morphine is almost completely absorbed (f = 1), so that loss in the gut is negligible.

However, the hepatic extraction ratio for morphine is 0.67, so (1 – ER) is 0.33. The bioavailability

of morphine is therefore expected to be about 33%, which is close to the observed value (Table 3–

1).

Rate of Absorption

The distinction between rate and extent of absorption is shown in Figure 3–4. The rate of absorption

is determined by the site of administration and the drug formulation. Both the rate of absorption and

the extent of input can influence the clinical effectiveness of a drug. For the three different dosage

forms depicted in Figure 3–4, there would be significant differences in the intensity of clinical

effect. Dosage form B would require twice the dose to attain blood concentrations equivalent to

those of dosage form A. Differences in rate of availability may become important for drugs given as

a single dose, such as a hypnotic used to induce sleep. In this case, drug from dosage form A would

reach its target concentration earlier than drug from dosage form C; concentrations from A would

also reach a higher level and remain above the target concentration for a longer period. In a multiple

dosing regimen, dosage forms A and C would yield the same average blood level concentrations,

although dosage form A would show somewhat greater maximum and lower minimum

concentrations.

The mechanism of drug absorption is said to be zero-order when the rate is independent of the

amount of drug remaining in the gut, eg, when it is determined by the rate of gastric emptying or by

a controlled-release drug formulation. In contrast, when the full dose is dissolved in gastrointestinal

fluids, the rate of absorption is usually proportional to the gastrointestinal concentration and is said

to be first-order.

Extraction Ratio & the First-Pass Effect

Systemic clearance is not affected by bioavailability. However, clearance can markedly affect the

extent of availability because it determines the extraction ratio (equation [8a]). Of course,

therapeutic blood concentrations may still be reached by the oral route of administration if larger

doses are given. However, in this case, the concentrations of the drug metabolites will be increased

significantly over those that would occur following intravenous administration. Lidocaine and

verapamil are both used to treat cardiac arrhythmias and have bioavailability less than 40%, but

lidocaine is never given orally because its metabolites are believed to contribute to central nervous

system toxicity. Other drugs that are highly extracted by the liver include isoniazid, morphine,

propranolol, verapamil, and several tricyclic antidepressants (Table 3–1).

Drugs with high extraction ratios will show marked variations in bioavailability between subjects

because of differences in hepatic function and blood flow. These differences can explain the marked

variation in drug concentrations that occurs among individuals given similar doses of highly

extracted drugs. For drugs that are highly extracted by the liver, shunting of blood past hepatic sites

of elimination will result in substantial increases in drug availability, whereas for drugs that are

poorly extracted by the liver (for which the difference between entering and exiting drug

concentration is small), shunting of blood past the liver will cause little change in availability.

Drugs in Table 3–1 that are poorly extracted by the liver include chlorpropamide, diazepam,

phenytoin, theophylline, tolbutamide, and warfarin.

Alternative Routes of Administration & the First-Pass Effect

There are several reasons for different routes of administration used in clinical medicine (Table 3–

3)—for convenience (eg, oral), to maximize concentration at the site of action and minimize it

elsewhere (eg, topical), to prolong the duration of drug absorption (eg, transdermal), or to avoid the

first-pass effect.

The hepatic first-pass effect can be avoided to a great extent by use of sublingual tablets and

transdermal preparations and to a lesser extent by use of rectal suppositories. Sublingual absorption

provides direct access to systemic—not portal—veins. The transdermal route offers the same

advantage. Drugs absorbed from suppositories in the lower rectum enter vessels that drain into the

inferior vena cava, thus bypassing the liver. However, suppositories tend to move upward in the

rectum into a region where veins that lead to the liver predominate. Thus, only about 50% of a

rectal dose can be assumed to bypass the liver.

Although drugs administered by inhalation bypass the hepatic first-pass effect, the lung may also

serve as a site of first-pass loss by excretion and possibly metabolism for drugs administered by

nongastrointestinal ("parenteral") routes.

The Time Course of Drug Effect

The principles of pharmacokinetics (discussed in this chapter) and those of pharmacodynamics

(discussed in Chapter 2: Drug Receptors & Pharmacodynamics; Holford & Sheiner, 1981) provide a

framework for understanding the time course of drug effect.

Immediate Effects

In the simplest case, drug effects are directly related to plasma concentrations, but this does not

necessarily mean that effects simply parallel the time course of concentrations. Because the

relationship between drug concentration and effect is not linear (recall the E

max

model described in

Chapter 2: Drug Receptors & Pharmacodynamics), the effect will not usually be linearly

proportional to the concentration.

Consider the effect of an angiotensin-converting enzyme (ACE) inhibitor, such as enalapril, on

plasma ACE. The half-life of enalapril is about 3 hours. After an oral dose of 10 mg, the peak

plasma concentration at 3 hours is about 64 ng/mL. Enalapril is usually given once a day, so seven

half-lives will elapse from the time of peak concentration to the end of the dosing interval. The

concentration of enalapril after each half-life and the corresponding extent of ACE inhibition are

shown in Figure 3–5. The extent of inhibition of ACE is calculated using the E

max

model, where

max

, the maximum extent of inhibition, is 100% and the EC

is about 1 ng/mL.

Figure 3–5.

Time course of angiotensin-converting enzyme (ACE) inhibitor concentrations and effects. The

black line shows the plasma enalapril concentrations in nanograms per milliliter after a single oral

dose. The colored line indicates the percentage inhibition of its target, ACE. Note the different

shapes of the concentration-time course (exponentially decreasing) and the effect-time course

(linearly decreasing in its central portion).

Note that plasma concentrations of enalapril change by a factor of 16 over the first 12 hours (four

half-lives) after the peak, but ACE inhibition has only decreased by 20%. Because the

concentrations over this time are so high in relation to the EC

, the effect on ACE is almost

constant. After 24 hours, ACE is still 33% inhibited. This explains why a drug with a short half-life

can be given once a day and still maintain its effect throughout the day. The key factor is a high

initial concentration in relation to the EC

. Even though the plasma concentration at 24 hours is

less than 1% of its peak, this low concentration is still half the EC

. This is very common for drugs

that act on enzymes (eg, ACE inhibitors) or compete at receptors (eg, propranolol).

When concentrations are in the range between one fourth and four times the EC

, the time course

of effect is essentially a linear function of time—13% of the effect is lost every half-life over this

concentration range. At concentrations below one fourth the EC

, the effect becomes almost

directly proportional to concentration and the time course of drug effect will follow the exponential

decline of concentration. It is only when the concentration is low in relation to the EC

that the

concept of a "half-life of drug effect" has any meaning.

Delayed Effects

Changes in drug effects are often delayed in relation to changes in plasma concentration. This delay

may reflect the time required for the drug to distribute from plasma to the site of action. This will be

the case for almost all drugs. The delay due to distribution is a pharmacokinetic phenomenon that

can account for delays of a few minutes. This distributional delay can account for the lag of effects

after rapid intravenous injection of central nervous system (CNS)–active agents such as thiopental.

A common reason for more delayed drug effects—especially those that take many hours or even

days to occur—is the slow turnover of a physiologic substance that is involved in the expression of

the drug effect (Jusko & Ko, 1994). For example, warfarin works as an anticoagulant by inhibiting

vitamin K epoxidase in the liver. This action of warfarin occurs rapidly, and inhibition of the

enzyme is closely related to plasma concentrations of warfarin. The clinical effect of warfarin, eg,

on the prothrombin time, reflects a decrease in the concentration of the prothrombin complex of

clotting factors (see Figure 34–7). Inhibition of vitamin K epoxidase decreases the synthesis of

these clotting factors, but the complex has a long half-life (about 14 hours), and it is this half-life

that determines how long it takes for the concentration of clotting factors to reach a new steady state

and for a drug effect to become manifest that reflects the warfarin plasma concentration.

Cumulative Effects

Some drug effects are more obviously related to a cumulative action than to a rapidly reversible

one. The renal toxicity of aminoglycoside antibiotics (eg, gentamicin) is greater when administered

as a constant infusion than with intermittent dosing. It is the accumulation of aminoglycoside in the

renal cortex that is thought to cause renal damage. Even though both dosing schemes produce the

same average steady-state concentration, the intermittent dosing scheme produces much higher peak

concentrations, which saturate an uptake mechanism into the cortex; thus, total aminoglycoside

accumulation is less. The difference in toxicity is a predictable consequence of the different patterns

of concentration and the saturable uptake mechanism.

The effect of many drugs used to treat cancer also reflects a cumulative action—eg, the extent of

binding of a drug to DNA is proportional to drug concentration and is usually irreversible. The

effect on tumor growth is therefore a consequence of cumulative exposure to the drug. Measures of

cumulative exposure, such as AUC, provide a means to individualize treatment (Evans et al, 1998).

The Target Concentration Approach to Designing a Rational Dosage Regimen

A rational dosage regimen is based on the assumption that there is a target concentration that will

produce the desired therapeutic effect. By considering the pharmacokinetic factors that determine

the dose-concentration relationship, it is possible to individualize the dose regimen to achieve the

target concentration. The effective concentration ranges shown in Table 3–1 are a guide to the

concentrations measured when patients are being effectively treated. The initial target concentration

should usually be chosen from the lower end of this range. In some cases, the target concentration

will also depend on the specific therapeutic objective—eg, the control of atrial fibrillation by

digoxin often requires a target concentration of 2 ng/mL, while heart failure is usually adequately

managed with a target concentration of 1 ng/mL.

Maintenance Dose

In most clinical situations, drugs are administered in such a way as to maintain a steady state of

drug in the body, ie, just enough drug is given in each dose to replace the drug eliminated since the

preceding dose. Thus, calculation of the appropriate maintenance dose is a primary goal. Clearance

is the most important pharmacokinetic term to be considered in defining a rational steady state drug

dosage regimen. At steady state, the dosing rate ("rate in") must equal the rate of elimination ("rate

out"). Substitution of the target concentration (TC) for concentration (C) in equation (4) predicts the

maintenance dosing rate:

Thus, if the desired target concentration is known, the clearance in that patient will determine the

dosing rate. If the drug is given by a route that has a bioavailability less than 100%, then the dosing

rate predicted by equation (9) must be modified. For oral dosing:

If intermittent doses are given, the maintenance dose is calculated from:

(See Example: Maintenance Dose Calculation.)

Note that the steady-state concentration achieved by continuous infusion or the average

concentration following intermittent dosing depends only on clearance. The volume of distribution

and the half-life need not be known in order to determine the average plasma concentration

expected from a given dosing rate or to predict the dosing rate for a desired target concentration.

Figure 3–6 shows that at different dosing intervals, the concentration time curves will have different

maximum and minimum values even though the average level will always be 10 mg/L.

Estimates of dosing rate and average steady-state concentrations, which may be calculated using

clearance, are independent of any specific pharmacokinetic model. In contrast, the determination of

maximum and minimum steady-state concentrations requires further assumptions about the

pharmacokinetic model. The accumulation factor (equation [7]) assumes that the drug follows a

one-compartment body model (Figure 3–2 B), and the peak concentration prediction assumes that

the absorption rate is much faster than the elimination rate. For the calculation of estimated

maximum and minimum concentrations in a clinical situation, these assumptions are usually

reasonable.

Example: Maintenance Dose Calculation

A target plasma theophylline concentration of 10 mg/L is desired to relieve acute bronchial asthma

in a patient. If the patient is a nonsmoker and otherwise normal except for asthma, we may use the

mean clearance given in Table 3–1, ie, 2.8 L/h/70 kg. Since the drug will be given as an intravenous

infusion, F = 1.

Therefore, in this patient, the proper infusion rate would be 28 mg/h/70 kg.

If the asthma attack is relieved, the clinician might want to maintain this plasma level using oral

theophylline, which might be given every 12 hours using an extended-release formulation to

approximate a continuous intravenous infusion. According to Table 3–1, F

oral

is 0.96. When the

dosing interval is 12 hours, the size of each maintenance dose would be:

A tablet or capsule size close to the ideal dose of 350 mg would then be prescribed at 12-hourly

intervals. If an 8-hour dosing interval was used, the ideal dose would be 233 mg; and if the drug

was given once a day, the dose would be 700 mg. In practice, F could be omitted from the

calculation since it is so close to 1.

Loading Dose

When the time to reach steady state is appreciable, as it is for drugs with long half-lives, it may be

desirable to administer a loading dose that promptly raises the concentration of drug in plasma to

the target concentration. In theory, only the amount of the loading dose need be computed—not the

rate of its administration—and, to a first approximation, this is so. The volume of distribution is the

proportionality factor that relates the total amount of drug in the body to the concentration in the

plasma (C

); if a loading dose is to achieve the target concentration, then from equation (1):

For the theophylline example given in Example: Maintenance Dose Calculation, the loading dose

would be 350 mg (35 L x 10 mg/L) for a 70 kg person. For most drugs, the loading dose can be

given as a single dose by the chosen route of administration.

Up to this point, we have ignored the fact that some drugs follow more complex multicompartment

pharmacokinetics, eg, the distribution process illustrated by the two-compartment model in Figure

3–2. This is justified in the great majority of cases. However, in some cases the distribution phase

may not be ignored, particularly in connection with the calculation of loading doses. If the rate of

absorption is rapid relative to distribution (this is always true for intravenous bolus administration),

the concentration of drug in plasma that results from an appropriate loading dose—calculated using

the apparent volume of distribution—can initially be considerably higher than desired. Severe

toxicity may occur, albeit transiently. This may be particularly important, for example, in the

administration of antiarrhythmic drugs such as lidocaine, where an almost immediate toxic response

may occur. Thus, while the estimation of the amount of a loading dose may be quite correct, the

rate of administration can sometimes be crucial in preventing excessive drug concentrations, and

slow administration of an intravenous drug (over minutes rather than seconds) is almost always

prudent practice. For intravenous doses of theophylline, initial injections should be given over a 20-

minute period to reduce the possibility of high plasma concentrations during the distribution phase.

When intermittent doses are given, the loading dose calculated from equation (12) will only reach

the average steady-state concentration and will not match the peak steady-state concentration (see

Figure 3–6). To match the peak steady-state concentration, the loading dose can be calculated from

equation (13):

Figure 3–6.

Relationship between frequency of dosing and maximum and minimum plasma concentrations

when a steady-state theophylline plasma level of 10 mg/L is desired. The smoothly rising line

(solid black) shows the plasma concentration achieved with an intravenous infusion of 28 mg/h.

The doses for 8-hourly administration (light color) are 224 mg; for 24-hourly administration (dark

color), 672 mg. In each of the 3 cases, the mean steady-state plasma concentration is 10 mg/L.

Therapeutic Drug Monitoring: Relating Pharmacokinetics & Pharmacodynamics

The basic principles outlined above can be applied to the interpretation of clinical drug

concentration measurements on the basis of three major pharmacokinetic variables: absorption,

clearance, and volume of distribution (and the derived variable, half-life); and two

pharmacodynamic variables: maximum effect attainable in the target tissue and the sensitivity of the

tissue to the drug. Diseases may modify all of these parameters, and the ability to predict the effect

of disease states on pharmacokinetic parameters is important in properly adjusting dosage in such

cases. (See The Target Concentration Strategy.)

The Target Concentration Strategy

Recognition of the essential role of concentration in linking pharmacokinetics and

pharmacodynamics leads naturally to the target concentration strategy. Pharmacodynamic principles

can be used to predict the concentration required to achieve a particular degree of therapeutic effect.

This target concentration can then be achieved by using pharmacokinetic principles to arrive at a

suitable dosing regimen (Holford, 1999). The target concentration strategy is a process for

optimizing the dose in an individual on the basis of a measured surrogate response such as drug

concentration:

1. Choose the target concentration, TC.

2. Predict volume of distribution (V

) and clearance (CL) based on standard population

values (eg, Table 3–1) with adjustments for factors such as weight and renal function.

3. Give a loading dose or maintenance dose calculated from TC, V

, and CL.

4. Measure the patient's response and drug concentration.

5. Revise V

and/or CL based on the measured concentration.

6. Repeat steps 3–5, adjusting the predicted dose to achieve TC.

Pharmacokinetic Variables

Absorption

The amount of drug that enters the body depends on the patient's compliance with the prescribed

regimen and on the rate and extent of transfer from the site of administration to the blood.

Overdosage and underdosage relative to the prescribed dosage—both aspects of failure of

compliance—can frequently be detected by concentration measurements when gross deviations

from expected values are obtained. If compliance is found to be adequate, absorption abnormalities

in the small bowel may be the cause of abnormally low concentrations. Variations in the extent of

bioavailability are rarely caused by irregularities in the manufacture of the particular drug

formulation. More commonly, variations in bioavailability are due to metabolism during absorption.

Clearance

Abnormal clearance may be anticipated when there is major impairment of the function of the

kidney, liver, or heart. Creatinine clearance is a useful quantitative indicator of renal function.

Conversely, drug clearance may be a useful indicator of the functional consequences of heart,

kidney, or liver failure, often with greater precision than clinical findings or other laboratory tests.

For example, when renal function is changing rapidly, estimation of the clearance of

aminoglycoside antibiotics may be a more accurate indicator of glomerular filtration than serum

creatinine.

Hepatic disease has been shown to reduce the clearance and prolong the half-life of many drugs.

However, for many other drugs known to be eliminated by hepatic processes, no changes in

clearance or half-life have been noted with similar hepatic disease. This reflects the fact that hepatic

disease does not always affect the hepatic intrinsic clearance. At present, there is no reliable marker

of hepatic drug-metabolizing function that can be used to predict changes in liver clearance in a

manner analogous to the use of creatinine clearance as a marker of renal drug clearance.

Volume of Distribution

The apparent volume of distribution reflects a balance between binding to tissues, which decreases

plasma concentration and makes the apparent volume larger, and binding to plasma proteins, which

increases plasma concentration and makes the apparent volume smaller. Changes in either tissue or

plasma binding can change the apparent volume of distribution determined from plasma

concentration measurements. Older people have a relative decrease in skeletal muscle mass and

tend to have a smaller apparent volume of distribution of digoxin (which binds to muscle proteins).

The volume of distribution may be overestimated in obese patients if based on body weight and the

drug does not enter fatty tissues well, as is the case with digoxin. In contrast, theophylline has a

volume of distribution similar to that of total body water. Adipose tissue has almost as much water

in it as other tissues, so that the apparent total volume of distribution of theophylline is proportional

to body weight even in obese patients.

Abnormal accumulation of fluid—edema, ascites, pleural effusion—can markedly increase the

volume of distribution of drugs such as gentamicin that are hydrophilic and have small volumes of

distribution.

Half-Life

The differences between clearance and half-life are important in defining the underlying

mechanisms for the effect of a disease state on drug disposition. For example, the half-life of

diazepam increases with age. When clearance is related to age, it is found that clearance of this drug

does not change with age. The increasing half-life for diazepam actually results from changes in the

volume of distribution with age; the metabolic processes responsible for eliminating the drug are

fairly constant.

Pharmacodynamic Variables

Maximum Effect

All pharmacologic responses must have a maximum effect (E

max

). No matter how high the drug

concentration goes, a point will be reached beyond which no further increment in response is

achieved.

If increasing the dose in a particular patient does not lead to a further clinical response, it is possible

that the maximum effect has been reached. Recognition of maximum effect is helpful in avoiding

ineffectual increases of dose with the attendant risk of toxicity.

Sensitivity

The sensitivity of the target organ to drug concentration is reflected by the concentration required to

produce 50% of maximum effect, the EC

. Failure of response due to diminished sensitivity to the

drug can be detected by measuring—in a patient who is not getting better—drug concentrations that

are usually associated with therapeutic response. This may be a result of abnormal physiology—eg,

hyperkalemia diminishes responsiveness to digoxin—or drug antagonism—eg, calcium channel

blockers impair the inotropic response to digoxin.

Increased sensitivity to a drug is usually signaled by exaggerated responses to small or moderate

doses. The pharmacodynamic nature of this sensitivity can be confirmed by measuring drug

concentrations that are low in relation to the observed effect.

Interpretation of Drug Concentration Measurements

Clearance

Clearance is the single most important factor determining drug concentrations. The interpretation of

measurements of drug concentrations depends on a clear understanding of three factors that may

influence clearance: the dose, the organ blood flow, and the intrinsic function of the liver or

kidneys. Each of these factors should be considered when interpreting clearance estimated from a

drug concentration measurement. It must also be recognized that changes in protein binding may

lead the unwary to believe there is a change in clearance when in fact drug elimination is not altered

(see Plasma Protein Binding: Is It Important?). Factors affecting protein binding include the

following:

1. Albumin concentration: Drugs such as phenytoin, salicylates, and disopyramide are

extensively bound to plasma albumin. Albumin levels are low in many disease states, resulting in

lower total drug concentrations.

2. Alpha

-acid glycoprotein concentration:

-Acid glycoprotein is an important binding protein

with binding sites for drugs such as quinidine, lidocaine, and propranolol. It is increased in acute

inflammatory disorders and causes major changes in total plasma concentration of these drugs

even though drug elimination is unchanged.

3. Capacity-limited protein binding: The binding of drugs to plasma proteins is capacity-limited.

Therapeutic concentrations of salicylates and prednisolone show concentration-dependent protein

binding. Because unbound drug concentration is determined by dosing rate and clearance—

which is not altered, in the case of these low-extraction-ratio drugs, by protein binding—

increases in dosing rate will cause corresponding changes in the pharmacodynamically important

unbound concentration. Total drug concentration will increase less rapidly than the dosing rate

would suggest as protein binding approaches saturation at higher concentrations.

Plasma Protein Binding: Is It Important?

Plasma protein binding is often mentioned as a factor playing a role in pharmacokinetics,

pharmacodynamics, and drug interactions. However, there are no clinically relevant examples of

changes in drug disposition or effects that can be clearly ascribed to changes in plasma protein

binding (Benet & Hoener 2002). The idea that if a drug is displaced from plasma proteins it would

increase the unbound drug concentration and increase the drug effect and, perhaps, produce toxicity

seems a simple and obvious mechanism. Unfortunately, this simple theory, which is appropriate for

a test tube, does not work in the body, which is an open system capable of eliminating unbound

drug.

First, a seemingly dramatic change in the unbound fraction from 1% to 10% releases less than 5%

of the total amount of drug in the body into the unbound pool because less than one third of the

drug in the body is bound to plasma proteins even in the most extreme cases, eg, warfarin. Drug

displaced from plasma protein will of course distribute throughout the volume of distribution, so

that a 5% increase in the amount of unbound drug in the body produces at most a 5% increase in

pharmacologically active unbound drug at the site of action.

Second, when the amount of unbound drug in plasma increases, the rate of elimination will increase

(if unbound clearance is unchanged), and after four half-lives the unbound concentration will return

to its previous steady state value. When drug interactions associated with protein binding

displacement and clinically important effects have been studied, it has been found that the

displacing drug is also an inhibitor of clearance, and it is the change in clearance of the unbound

drug that is the relevant mechanism explaining the interaction.

The clinical importance of plasma protein binding is only to help interpretation of measured drug

concentrations. When plasma proteins are lower than normal, then total drug concentrations will be

lower but unbound concentrations will not be affected.

Dosing History

An accurate dosing history is essential if one is to obtain maximum value from a drug concentration

measurement. In fact, if the dosing history is unknown or incomplete, a drug concentration

measurement loses all predictive value.

Timing of Samples for Concentration Measurement

Information about the rate and extent of drug absorption in a particular patient is rarely of great

clinical importance. However, absorption usually occurs during the first 2 hours after a drug dose

and varies according to food intake, posture, and activity. Therefore, it is important to avoid

drawing blood until absorption is complete (about 2 hours after an oral dose). Attempts to measure

peak concentrations early after oral dosing are usually unsuccessful and compromise the validity of

the measurement, because one cannot be certain that absorption is complete.

Some drugs such as digoxin and lithium take several hours to distribute to tissues. Digoxin samples

should be taken at least 6 hours after the last dose and lithium just before the next dose (usually 24

hours after the last dose). Aminoglycosides distribute quite rapidly, but it is still prudent to wait 1

hour after giving the dose before taking a sample.

Clearance is readily estimated from the dosing rate and mean steady-state concentration. Blood

samples should be appropriately timed to estimate steady-state concentration. Provided steady state

has been approached (at least three half-lives of constant dosing), a sample obtained near the

midpoint of the dosing interval will usually be close to the mean steady-state concentration.

Initial Predictions of Volume of Distribution & Clearance

Volume of Distribution

Volume of distribution is commonly calculated for a particular patient using body weight (70 kg

body weight is assumed for the values in Table 3–1). If a patient is obese, drugs that do not readily

penetrate fat (eg, gentamicin and digoxin) should have their volumes calculated from ideal body

weight as shown below:

Patients with edema, ascites, or pleural effusions offer a larger volume of distribution to the

aminoglycoside antibiotics (eg, gentamicin) than is predicted by body weight. In such patients, the

weight should be corrected as follows: Subtract an estimate of the weight of the excess fluid

accumulation from the measured weight. Use the resultant "normal" body weight to calculate the

normal volume of distribution. Finally, this normal volume should be increased by 1 L for each

estimated kilogram of excess fluid. This correction is important because of the relatively small

volumes of distribution of these water-soluble drugs.

Clearance

Drugs cleared by the renal route often require adjustment of clearance in proportion to renal

function. This can be conveniently estimated from the creatinine clearance, calculated from a single

serum creatinine measurement and the predicted creatinine production rate.

The predicted creatinine production rate in women is 85% of the calculated value, because they

have a smaller muscle mass per kilogram and it is muscle mass that determines creatinine

production. Muscle mass as a fraction of body weight decreases with age, which is why age appears

in the Cockcroft-Gault equation, given in Chapter 61: Special Aspects of Geriatric Pharmacology.

The decrease of renal function with age is independent of the decrease in creatinine production.

Because of the difficulty of obtaining complete urine collections, creatinine clearance calculated in

this way is at least as reliable as estimates based on urine collections. Ideal body weight should be

used for obese patients, and correction should be made for muscle wasting in severely ill patients.

Revising Individual Estimates of Volume of Distribution & Clearance

The commonsense approach to the interpretation of drug concentrations compares predictions of

pharmacokinetic parameters and expected concentrations to measured values. If measured

concentrations differ by more than 20% from predicted values, revised estimates of V

or CL for

that patient should be calculated using equation (1) or equation (2). If the change calculated is more

than a 100% increase or 50% decrease in either V

or CL, the assumptions made about the timing of

the sample and the dosing history should be critically examined.

For example, if a patient is taking 0.25 mg of digoxin a day, a clinician may expect the digoxin

concentration to be about 1 ng/mL. This is based on typical values for bioavailability of 70% and

total clearance of about 7 L/h (CL

renal

4 L/h, CL

nonrenal

3 L/h). If the patient has heart failure, the

nonrenal (hepatic) clearance might be halved because of hepatic congestion and hypoxia, so the

expected clearance would become 5.5 L/h. The concentration is then expected to be about 1.3

ng/mL. Suppose that the concentration actually measured is 2 ng/mL. Common sense would

suggest halving the daily dose to achieve a target concentration of 1 ng/mL. This approach implies a

revised clearance of 3.5 L/h. The smaller clearance compared with the expected value of 5.5 L/h

may reflect additional renal functional impairment due to heart failure.

This technique will often be misleading if steady state has not been reached. At least a week of

regular dosing (three to four half-lives) must elapse before the implicit method will be reliable.

Chapter 4. Drug Biotransformation

Drug Biotransformation: Introduction

Humans are exposed daily to a wide variety of foreign compounds called xenobiotics—substances

absorbed across the lungs or skin or, more commonly, ingested either unintentionally as compounds

present in food and drink or deliberately as drugs for therapeutic or "recreational" purposes.

Exposure to environmental xenobiotics may be inadvertent and accidental or—when they are

present as components of air, water, and food—inescapable. Some xenobiotics are innocuous, but

many can provoke biologic responses. Such biologic responses often depend on conversion of the

absorbed substance into an active metabolite. The discussion that follows is applicable to

xenobiotics in general (including drugs) and to some extent to endogenous compounds.

Why Is Drug Biotransformation Necessary?

Renal excretion plays a pivotal role in terminating the biologic activity of some drugs, particularly

those that have small molecular volumes or possess polar characteristics such as functional groups

that are fully ionized at physiologic pH. However, many drugs do not possess such physicochemical

properties. Pharmacologically active organic molecules tend to be lipophilic and remain un-ionized

or only partially ionized at physiologic pH. They are often strongly bound to plasma proteins. Such

substances are not readily filtered at the glomerulus. The lipophilic nature of renal tubular

membranes also facilitates the reabsorption of hydrophobic compounds following their glomerular

filtration. Consequently, most drugs would have a prolonged duration of action if termination of

their action depended solely on renal excretion.

An alternative process that may lead to the termination or alteration of biologic activity is

metabolism. In general, lipophilic xenobiotics are transformed to more polar and hence more

readily excretable products. The role metabolism may play in the inactivation of lipid-soluble drugs

can be quite dramatic. For example, lipophilic barbiturates such as thiopental and pentobarbital

would have extremely long half-lives if it were not for their metabolic conversion to more water-

soluble compounds.

Metabolic products are often less pharmacodynamically active than the parent drug and may even

be inactive. However, some biotransformation products have enhanced activity or toxic properties.

It is noteworthy that the synthesis of endogenous substrates such as steroid hormones, cholesterol,

active vitamin D congeners and bile acids involves many pathways catalyzed by enzymes

associated with the metabolism of xenobiotics. Finally, drug-metabolizing enzymes have been

exploited in the design of pharmacologically inactive prodrugs that are converted to active

molecules in the body.

Where Do Drug Biotransformations Occur?

Although every tissue has some ability to metabolize drugs, the liver is the principal organ of drug

metabolism. Other tissues that display considerable activity include the gastrointestinal tract, the

lungs, the skin, and the kidneys. Following oral administration, many drugs (eg, isoproterenol,

meperidine, pentazocine, morphine) are absorbed intact from the small intestine and transported

first via the portal system to the liver, where they undergo extensive metabolism. This process has

been called a first-pass effect (see Chapter 3: Pharmacokinetics & Pharmacodynamics: Rational

Dosing & the Time Course of Drug Action). Some orally administered drugs (eg, clonazepam,

chlorpromazine) are more extensively metabolized in the intestine than in the liver. Thus, intestinal

metabolism may contribute to the overall first-pass effect. First-pass effects may so greatly limit the

bioavailability of orally administered drugs that alternative routes of administration must be used to

achieve therapeutically effective blood levels. The lower gut harbors intestinal microorganisms that

are capable of many biotransformation reactions. In addition, drugs may be metabolized by gastric

acid (eg, penicillin), by digestive enzymes (eg, polypeptides such as insulin), or by enzymes in the

wall of the intestine (eg, sympathomimetic catecholamines).

Although drug biotransformation in vivo can occur by spontaneous, noncatalyzed chemical

reactions, the vast majority of transformations are catalyzed by specific cellular enzymes. At the

subcellular level, these enzymes may be located in the endoplasmic reticulum, mitochondria,

cytosol, lysosomes, or even the nuclear envelope or plasma membrane.

Microsomal Mixed Function Oxidase System & Phase I Reactions

Many drug-metabolizing enzymes are located in the lipophilic membranes of the endoplasmic

reticulum of the liver and other tissues. When these lamellar membranes are isolated by

homogenization and fractionation of the cell, they re-form into vesicles called microsomes.

Microsomes retain most of the morphologic and functional characteristics of the intact membranes,

including the rough and smooth surface features of the rough (ribosome-studded) and smooth (no

ribosomes) endoplasmic reticulum. Whereas the rough microsomes tend to be dedicated to protein

synthesis, the smooth microsomes are relatively rich in enzymes responsible for oxidative drug

metabolism. In particular, they contain the important class of enzymes known as the mixed

function oxidases (MFOs), or monooxygenases. The activity of these enzymes requires both a

reducing agent (NADPH) and molecular oxygen; in a typical reaction, one molecule of oxygen is

consumed (reduced) per substrate molecule, with one oxygen atom appearing in the product and the

other in the form of water.

In this oxidation-reduction process, two microsomal enzymes play a key role. The first of these is a

flavoprotein, NADPH-cytochrome P450 reductase. One mole of this enzyme contains 1 mol each

of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The second microsomal

enzyme is a hemoprotein called cytochrome P450 that serves as the terminal oxidase. In fact, the

microsomal membrane harbors multiple forms of this hemoprotein, and this multiplicity is

increased by repeated administration of exogenous chemicals (see below). The name cytochrome

P450 (abbreviated as CYP or P450) is derived from the spectral properties of this hemoprotein. In

its reduced (ferrous) form, it binds carbon monoxide to give a complex that absorbs light maximally

at 450 nm. The relative abundance of P450, compared with that of the reductase in the liver,

contributes to making P450 heme reduction a rate-limiting step in hepatic drug oxidations.

Microsomal drug oxidations require P450, P450 reductase, NADPH, and molecular oxygen. A

simplified scheme of the oxidative cycle is presented in Figure 4–3. Briefly, oxidized (Fe

) P450

combines with a drug substrate to form a binary complex (step 1). NADPH donates an electron to

the flavoprotein reductase, which in turn reduces the oxidized P450-drug complex (step 2). A

second electron is introduced from NADPH via the same flavoprotein reductase, which serves to

reduce molecular oxygen and to form an "activated oxygen"- P450-substrate complex (step 3). This

complex in turn transfers activated oxygen to the drug substrate to form the oxidized product (step

4).

Figure 4–3.

Cytochrome P450 cycle in drug oxidations. (R-H, parent drug; R-OH, oxidized metabolite; e

electron.)

The potent oxidizing properties of this activated oxygen permit oxidation of a large number of

substrates. Substrate specificity is very low for this enzyme complex. High solubility in lipids is the

only common structural feature of the wide variety of structurally unrelated drugs and chemicals

that serve as substrates in this system (Table 4–1).

Table 4–1. Phase I Reactions.

Enzyme Induction

Some of these chemically dissimilar drug substrates, on repeated administration, "induce" P450 by

enhancing the rate of its synthesis or reducing its rate of degradation. Induction results in an

acceleration of substrate metabolism and usually in a decrease in the pharmacologic action of the

inducer and also of coadministered drugs. However, in the case of drugs metabolically transformed

to reactive metabolites, enzyme induction may exacerbate metabolite-mediated toxicity.

Various substrates appear to induce P450 isoforms having different molecular masses and

exhibiting different substrate specificities and immunochemical and spectral characteristics. The

isoforms that have been most extensively studied include CYP2B1 (formerly P450b), induced by

phenobarbital treatment; CYP1A1 (P

450 or P448), induced by polycyclic aromatic hydrocarbons

("PAHs" such as benzo[a]pyrene and 3-methylcholanthrene); CYPs3A (including CYPs 3A4 and

3A5, the major human liver isoforms) induced by glucocorticoids, macrolide antibiotics,

anticonvulsants, and some steroids. Chronic administration of isoniazid or ethanol induces a

different isoform, CYP2E1, that oxidizes ethanol and activates carcinogenic nitrosamines. The

VLDL-lowering drug clofibrate induces other distinct enzymes of the CYP4A class that are

responsible for -hydroxylation of several fatty acids, leukotrienes, and prostaglandins.

Environmental pollutants are also capable of inducing P450 enzymes. As noted above, exposure to

benzo[a]pyrene and other polycyclic aromatic hydrocarbons, which are present in tobacco smoke,

charcoal-broiled meat, and other organic pyrolysis products, is known to induce CYP1A enzymes

and to alter the rates of drug metabolism. Other environmental chemicals known to induce specific

P450s include the polychlorinated biphenyls (PCBs), which were used widely in industry as

insulating materials and plasticizers, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin, TCDD), a

trace byproduct of the chemical synthesis of the defoliant 2,4,5-T (see Chapter 57: Introduction to

Toxicology: Occupational & Environmental).

Increased P450 synthesis requires enhanced transcription and translation. A cytoplasmic receptor

(termed AhR) for polycyclic aromatic hydrocarbons (eg, benzo[a]pyrene, dioxin) has been

identified, and the translocation of the inducer-receptor complex into the nucleus and subsequent

activation of regulatory elements of genes have been documented. A pregnane X receptor (PXR), a

member of the steroid-retinoid-thyroid hormone receptor family, has recently been shown to

mediate CYP3A induction by various chemicals (dexamethasone, rifampin) in the liver and

intestinal mucosa. A similar receptor, the constitutive androstane receptor (CAR) has been

identified for the phenobarbital class of inducers (Sueyoshi, 2001; Willson, 2002).

P450 enzymes may also be induced by "substrate stabilization," ie, decreased degradation, as is the

case with troleandomycin- or clotrimazole-mediated induction of CYP3A enzymes and the ethanol-

mediated induction of CYP2E1.

Enzyme Inhibition

Certain drug substrates may inhibit cytochrome P450 enzyme activity. Imidazole-containing drugs

such as cimetidine and ketoconazole bind tightly to the P450 heme iron and effectively reduce the

metabolism of endogenous substrates (testosterone) or other coadministered drugs through

competitive inhibition. However, macrolide antibiotics such as troleandomycin, erythromycin, and

other erythromycin derivatives are metabolized, apparently by CYP3A, to metabolites that complex

the cytochrome heme-iron and render it catalytically inactive. Another compound that acts through

this mechanism is the well-known inhibitor proadifen (SKF-525-A), which binds tightly to the

heme-iron and quasi-irreversibly inactivates the enzyme, thereby inhibiting the metabolism of

potential substrates.

Some substrates irreversibly inhibit P450s via covalent interaction of a metabolically generated

reactive intermediate that may react with the P450 apoprotein or heme moiety or even cause the

heme to fragment and irreversibly modify the apoprotein. The antibiotic chloramphenicol is

metabolized by CYP2B1 to a species that modifies its protein and thus also inactivates the enzyme.

A growing list of "suicide inhibitors"—inactivators that attack the heme or the protein moiety—

includes the steroids ethinyl estradiol, norethindrone, and spironolactone; the anesthetic agent

fluroxene; the barbiturate allobarbital; the analgesic sedatives allylisopropylacetylurea,

diethylpentenamide, and ethchlorvynol; the solvent carbon disulfide; and propylthiouracil. On the

other hand, the barbiturate secobarbital is found to inactivate CYP2B1 by modification of both its

heme and protein moieties.

Human Liver P450 Enzymes

Immunoblotting analyses—coupled with the use of relatively selective functional markers and

selective P450 inhibitors—have identified numerous P450 isoforms (CYPs 1A2, 2A6, 2B6, 2C9,

2C19, 2D6, 2E1, 3A4, 3A5, 4A11 and 7) in human liver microsomal preparations. Of these, CYPs

1A2, 2A6, 2C9, 2D6, 2E1, and 3A4 appear to be the major forms, accounting for approximately,

12, 4, 20, 4, 6, and 28 percent, respectively, of the total human liver P450 content. Together they are

responsible for catalyzing the bulk of the hepatic drug and xenobiotic metabolism (Table 4–2). It is

noteworthy that CYP3A4 alone is responsible for metabolism of more than 50% of the clinically

prescribed drugs metabolized by the liver. The involvement of individual P450s in the metabolism

of a given drug may be screened in vitro by means of selective functional markers, selective

chemical P450 inhibitors, and anti-P450 antibodies. In vivo, such screening may be accomplished

by means of relatively selective noninvasive markers, which include breath tests or urinary analyses

of specific metabolites after administration of a P450-selective substrate probe.

Table 4–2. Human Liver P450s (CYPs), and Some of the Drugs Metabolized (Substrates),

Inducers, and Drugs Used for Screening (Noninvasive Markers).

CYP Substrates Inducers Noninvasive Markers

1A2 Acetaminophen, antipyrine,

caffeine, clomipramine, phenacetin,

tamoxifen, theophylline, warfarin

Smoking, charcoal-

broiled foods,

cruciferous

vegetables,

omeprazole

Caffeine

2A6 Coumarin Coumarin

2B6 Artemisinin, bupropion,

cyclophosphamide, S-

mephobarbital, S-mephenytoin (N-

demethylation to nirvanol),

propofol, selegiline, sertraline

Phenobarbital,

cyclophosphamide

S-Mephenytoin

2C9 Hexobarbital, ibuprofen, phenytoin,

tolbutamide, trimethadione,

sulfaphenazole, S-warfarin,

Barbiturates, rifampin Tolbutamide,warfarin

ticrynafen

2C19 Diazepam, S-mephenytoin,

naproxen, nirvanol, omeprazole,

propranolol

Barbiturates, rifampin S-Mephenytoin

2D6 Bufuralol, bupranolol,

clomipramine, clozapine, codeine,

debrisoquin, dextromethorphan,

encainide, flecainide, fluoxetine,

guanoxan, haloperidol,

hydrocodone, 4-methoxy-

amphetamine, metoprolol,

mexiletine, oxycodone, paroxetine,

phenformin, propafenone,

propoxyphene, risperidone,

selegiline (deprenyl), sparteine,

thioridazine, timolol, tricyclic

antidepressants

None known Debrisoquin,dextromethorphan

2E1 Acetaminophen, chlorzoxazone,

enflurane, halothane, ethanol (a

minor pathway)

Ethanol, isoniazid Chlorzoxazone

3A4 Acetaminophen, alfentanil,

amiodarone, astemizole, cocaine,

cortisol, cyclosporine, dapsone,

diazepam, dihydroergotamine,

dihydropyridines, diltiazem, ethinyl

estradiol, gestodene, indinavir,

lidocaine, lovastatin, macrolides,

methadone, miconazole,

midazolam, mifepristone (RU 486),

paclitaxel, progesterone, quinidine,

rapamycin, ritonavir, saquinavir,

spironolactone, sulfamethoxazole,

sufentanil, tacrolimus, tamoxifen,

terfenadine, testosterone,

tetrahydro-cannabinol, triazolam,

troleandomycin, verapamil

Barbiturates,

carbamazepine,

macrolides,

glucocorticoids,

pioglitazone,

phenytoin, rifampin

Erythromycin, 6 -

hydroxycortisol

Phase II Reactions

Parent drugs or their phase I metabolites that contain suitable chemical groups often undergo

coupling or conjugation reactions with an endogenous substance to yield drug conjugates (Table 4–

3). In general, conjugates are polar molecules that are readily excreted and often inactive. Conjugate

formation involves high-energy intermediates and specific transfer enzymes. Such enzymes

(transferases) may be located in microsomes or in the cytosol. They catalyze the coupling of an

activated endogenous substance (such as the uridine 5'-diphosphate [UDP] derivative of glucuronic

acid) with a drug (or endogenous compound), or of an activated drug (such as the S-CoA derivative

of benzoic acid) with an endogenous substrate. Because the endogenous substrates originate in the

diet, nutrition plays a critical role in the regulation of drug conjugations.

Table 4–3. Phase II Reactions.

Type of

Conjugation

Endogenous

Reactant

Transferase

(Location)

Types of

Substrates

Examples

Glucuronidation UDP glucuronic

acid

UDP

glucuronosyl-

transferase

(microsomes)

Phenols, alcohols,

carboxylic acids,

hydroxylamines,

sulfonamides

Nitrophenol,

morphine,

acetaminophen,

diazepam, N-

hydroxydapsone,

sulfathiazole,

meprobamate,

digitoxin, digoxin

Acetylation Acetyl-CoA N-

Acetyltransferase

(cytosol)

Amines Sulfonamides,

isoniazid,

clonazepam,

dapsone, mescaline

Glutathione

conjugation

Glutathione GSH-S-transferase

(cytosol,

microsomes)

Epoxides, arene

oxides, nitro

groups,

hydroxylamines

Ethacrynic acid,

bromobenzene

Glycine

conjugation

Glycine Acyl-CoA

glycinetransferase

(mitochondria)

Acyl-CoA

derivatives of

carboxylic acids

Salicylic acid,

benzoic acid,

nicotinic acid,

cinnamic acid, cholic

acid, deoxycholic

acid

Sulfate

conjugation

Phosphoadenosyl

phosphosulfate

Sulfotransferase

(cytosol)

Phenols, alcohols,

aromatic amines

Estrone, aniline,

phenol, 3-hydroxy-

coumarin,

acetaminophen,

methyldopa

Methylation S-adenosyl-

methionine

Transmethylases

(cytosol)

Catecholamines,

phenols, amines

Dopamine,

epinephrine,

pyridine, histamine,

thiouracil

Epoxide hydrolase

(microsomes)

Arene oxides, cis-

disubstituted and

monosubstituted

oxiranes

Benzopyrene 7,8-

epoxide, styrene 1,2-

oxide,

carbamazepine

epoxide

Water

conjugation

Water

(cytosol) Alkene oxides,

fatty acid epoxides

Leukotriene A

Drug conjugations were once believed to represent terminal inactivation events and as such have

been viewed as "true detoxification" reactions. However, this concept must be modified, since it is

now known that certain conjugation reactions (acyl glucuronidation of nonsteroidal anti-

inflammatory drugs, O-sulfation of N-hydroxyacetylaminofluorene, and N-acetylation of isoniazid)

may lead to the formation of reactive species responsible for the hepatotoxicity of the drugs.

Furthermore, sulfation is known to activate the orally active prodrug minoxidil into a very

efficacious vasodilator.

Metabolism of Drugs to Toxic Products

It has become evident that metabolism of drugs and other foreign chemicals may not always be an

innocuous biochemical event leading to detoxification and elimination of the compound. Indeed,

several compounds have been shown to be metabolically transformed to reactive intermediates that

are toxic to various organs. Such toxic reactions may not be apparent at low levels of exposure to

parent compounds when alternative detoxification mechanisms are not yet overwhelmed or

compromised and the availability of endogenous detoxifying cosubstrates (glutathione [GSH],

glucuronic acid, sulfate) is not limited. However, when these resources are exhausted, the toxic

pathway may prevail, resulting in overt organ toxicity or carcinogenesis. The number of specific

examples of such drug-induced toxicity is expanding rapidly. An example is acetaminophen

(paracetamol)-induced hepatotoxicity (Figure 4–4). This analgesic antipyretic drug is quite safe in

therapeutic doses (1.2 g/d for an adult). It normally undergoes glucuronidation and sulfation to the

corresponding conjugates, which together comprise 95% of the total excreted metabolites. The

alternative P450-dependent glutathione conjugation pathway accounts for the remaining 5%. When

acetaminophen intake far exceeds therapeutic doses, the glucuronidation and sulfation pathways are

saturated, and the P450-dependent pathway becomes increasingly important. Little or no

hepatotoxicity results as long as glutathione is available for conjugation. However, with time,

hepatic glutathione is depleted faster than it can be regenerated, and a reactive and toxic metabolite

accumulates. In the absence of intracellular nucleophiles such as glutathione, this reactive

metabolite (N-acetylbenzoiminoquinone) reacts with nucleophilic groups of cellular proteins,

resulting in hepatotoxicity (Figure 4–4).

Figure 4–4.

Metabolism of acetaminophen (Ac) to hepatotoxic metabolites. (GSH, glutathione; GS,

glutathione moiety; Ac*, reactive intermediate.)

The chemical and toxicologic characterization of the electrophilic nature of the reactive

acetaminophen metabolite has led to the development of effective antidotes—cysteamine and N-

acetylcysteine. Administration of N-acetylcysteine (the safer of the two) within 8–16 hours

following acetaminophen overdosage has been shown to protect victims from fulminant

hepatotoxicity and death (see Chapter 59: Management of the Poisoned Patient).

Clinical Relevance of Drug Metabolism

The dose and the frequency of administration required to achieve effective therapeutic blood and

tissue levels vary in different patients because of individual differences in drug distribution and

rates of drug metabolism and elimination. These differences are determined by genetic factors and

nongenetic variables such as age, sex, liver size, liver function, circadian rhythm, body temperature,

and nutritional and environmental factors such as concomitant exposure to inducers or inhibitors of

drug metabolism. The discussion that follows summarizes the most important of these variables.

Individual Differences

Individual differences in metabolic rate depend on the nature of the drug itself. Thus, within the

same population, steady-state plasma levels may reflect a 30-fold variation in the metabolism of one

drug and only a twofold variation in the metabolism of another.

Genetic Factors

Genetic factors that influence enzyme levels account for some of these differences. Succinylcholine,

for example, is metabolized only half as rapidly in persons with genetically determined defects in

pseudocholinesterase as in persons with normally functioning pseudocholinesterase. Analogous

pharmacogenetic differences are seen in the acetylation of isoniazid (Figure 4–5) and the

hydroxylation of warfarin. The defect in slow acetylators (of isoniazid and similar amines) appears

to be caused by the synthesis of less of the enzyme rather than of an abnormal form of it. Inherited

as an autosomal recessive trait, the slow acetylator phenotype occurs in about 50% of blacks and

whites in the USA, more frequently in Europeans living in high northern latitudes, and much less

commonly in Asians and Inuits (Eskimos). Similarly, genetically determined defects in the

oxidative metabolism of debrisoquin, phenacetin, guanoxan, sparteine, phenformin, warfarin and

others have been reported (Table 4–4). The defects are apparently transmitted as autosomal

recessive traits and may be expressed at any one of the multiple metabolic transformations that a

chemical might undergo.

Figure 4–5.

Genetic polymorphism in drug metabolism. The graph shows the distribution of plasma

concentrations of isoniazid in 267 individuals 6 hours after an oral dose of 9.8 mg/kg. This

distribution is clearly bimodal. Individuals with a plasma concentration greater than 2.5 mg/mL at

6 hours are considered slow acetylators. (Redrawn, with permission, from Evans DAP, Manley

KA, McKusick VA: Genetic control of isoniazid metabolism in man. Br Med J 1960;2:485.)

Table 4–4. Some Examples of Genetic Polymorphisms in Drug Metabolism.

Defect Drug and Therapeutic Use Clinical Consequences

Oxidation Bufuralol ( -adrenoceptor blocker) Exacerbation of -blockade,

nausea

Oxidation Codeine (analgesic)

Reduced analgesia

Oxidation Debrisoquin (antihypertensive) Orthostatic hypotension

N-Demethylation Ethanol Facial flushing, cardiovascular

symptoms

Oxidation Ethanol Facial flushing, cardiovascular

symptoms

N-Acetylation Hydralazine (antihypertensive) Lupus erythematosus-like

syndrome

N-Acetylation Isoniazid (antitubercular) Peripheral neuropathy

Oxidation Mephenytoin (antiepileptic) Overdose toxicity

Thiopurine methyl-

transferase

Mercaptopurines (antileukemic) Myelotoxicity

Oxidation Nicotine (stimulant) Lesser addiction

Oxidation Nortriptyline (antidepressant) Toxicity

O-Demethylation Omeprazole (antiulcer) Increased therapeutic efficacy

Oxidation Sparteine Oxytocic symptoms

Ester hydrolysis Succinylcholine (neuromuscular

blocker)

Prolonged apnea

Oxidation S-warfarin (anticoagulant) Bleeding

Oxidation Tolbutamide (hypoglycemic) Cardiotoxicity

Observed or predictable.

Prodrug

Of the several recognized genetic variations of drug metabolism polymorphisms, three have been

particularly well characterized and afford some insight into possible underlying mechanisms. First

is the debrisoquin-sparteine oxidation type of polymorphism, which apparently occurs in 3–10% of

whites and is inherited as an autosomal recessive trait. In affected individuals, the CYP2D6-

dependent oxidations of debrisoquin and other drugs (see Table 4–2) are impaired. These defects in

oxidative drug metabolism are probably coinherited. The precise molecular basis for the defect

appears to be faulty expression of the P450 protein, resulting in little or no isoform-catalyzed drug

metabolism. More recently, however, another polymorphic genotype has been reported that results

in ultrarapid metabolism of relevant drugs due to the presence of 2D6 allelic variants with up to 13

gene copies in tandem. This genotype is most common in Ethiopians and Saudi Arabians,

populations that display it in up to one third of individuals. As a result, these subjects require

twofold to threefold higher daily doses of nortriptyline (a 2D6 substrate) to achieve therapeutic

plasma levels. Conversely, in these ultrarapidly metabolizing populations, the prodrug codeine

(another 2D6 substrate) is metabolized much faster to morphine, often resulting in undesirable side

effects of morphine such as severe abdominal pain.

A second well-studied genetic drug polymorphism involves the stereoselective aromatic (4)-

hydroxylation of the anticonvulsant mephenytoin, catalyzed by CYP2C19. This polymorphism,

which is also inherited as an autosomal recessive trait, occurs in 3–5% of Caucasians and 18–23%

of Japanese populations. It is genetically independent of the debrisoquin-sparteine polymorphism.

In normal "extensive metabolizers," (S)-mephenytoin is extensively hydroxylated by CYP2C19 at

the 4 position of the phenyl ring before its glucuronidation and rapid excretion in the urine, whereas

(R)-mephenytoin is slowly N-demethylated to nirvanol, an active metabolite. "Poor metabolizers,"

however, appear to totally lack the stereospecific (S)-mephenytoin hydroxylase activity, so both

(S)- and (R)-mephenytoin enantiomers are N-demethylated to nirvanol, which accumulates in much

higher concentrations. Thus, poor metabolizers of mephenytoin show signs of profound sedation

and ataxia after doses of the drug that are well tolerated by normal metabolizers. The molecular

basis for this defect is a single base pair mutation in exon 5 of the CYP2C19 gene that creates an

aberrant splice site, a correspondingly altered reading frame of the mRNA, and, finally, a truncated

nonfunctional protein. It is clinically important to recognize that the safety of a drug may be

severely reduced in individuals who are poor metabolizers.

The third genetic polymorphism recently characterized is that of CYP2C9. Two well-characterized

variants of this enzyme exist, each with amino acid mutations that result in altered metabolism:

CYP2C9*2 allele encodes an Arg144Cys mutation, exhibiting impaired functional interactions with

P450 reductase. The other allelic variant, CYP2C9*3, encodes an enzyme with an Ile359Leu

mutation that has lowered affinity for many substrates. Consequently, individuals displaying the

CYP2C9*3 phenotype have greatly reduced tolerance for the anticoagulant warfarin. The warfarin

clearance in CYP2C9*3-homozygous individuals is only 10% of normal values, and these people

can tolerate much smaller daily doses of the drug than those who are homozygous for the normal

wild type allele. These individuals also have a much higher risk of adverse effects with warfarin

(eg, bleeding) and with other CYP2C9 substrates such as phenytoin, losartan, tolbutamide, and

some NSAIDs.

Allelic variants of CYP3A4 have also been reported but their contribution to its well-known

interindividual variability in drug metabolism apparently is limited. On the other hand, the

expression of CYP3A5, another human liver isoform, is markedly polymorphic, ranging from 0% to

100% of the total hepatic CYP3A content. This CYP3A5 protein polymorphism is now known to

result from a single nucleotide polymorphism (SNIP) within intron 3 which enables normally

spliced CYP3A5 transcripts in 5% of Caucasians, 29% of Japanese, 27% of Chinese, 30% of

Koreans, and 73% of African Americans. Thus, it can significantly contribute to interindividual

differences in the metabolism of preferential CYP3A5 substrates such as midazolam.

Additional genetic polymorphisms in drug metabolism that are inherited independently from those

already described are being discovered. Studies of theophylline metabolism in monozygotic and

dizygotic twins that included pedigree analysis of various families have revealed that a distinct

polymorphism may exist for this drug and may be inherited as a recessive genetic trait. Genetic

drug metabolism polymorphisms also appear to occur for aminopyrine and carbocysteine

oxidations. Regularly updated information on human P450-polymorphisms is available at

http://www.imm.ki.se/CYPalleles/.

Although genetic polymorphisms in drug oxidations often involve specific P450 enzymes, such

genetic variations can occur at other sites. The recent descriptions of a polymorphism in the

oxidation of trimethylamine, believed to be metabolized largely by the flavin monooxygenase

(Ziegler's enzyme), suggest that genetic variants of other non-P450-dependent oxidative enzymes

may also contribute to such polymorphisms.

Diet & Environmental Factors

Diet and environmental factors also contribute to individual variations in drug metabolism.

Charcoal-broiled foods and cruciferous vegetables are known to induce CYP1A enzymes, whereas

grapefruit juice is known to inhibit the CYP3A metabolism of coadministered drug substrates.

Cigarette smokers metabolize some drugs more rapidly than nonsmokers because of enzyme

induction (see above). Industrial workers exposed to some pesticides metabolize certain drugs more

rapidly than nonexposed individuals. Such differences make it difficult to determine effective and

safe doses of drugs that have narrow therapeutic indices.

Age & Sex

Increased susceptibility to the pharmacologic or toxic activity of drugs has been reported in very

young and old patients compared with young adults (see Chapters 60 and 61). Although this may

reflect differences in absorption, distribution, and elimination, differences in drug metabolism also

plays a role. Slower metabolism could be due to reduced activity of metabolic enzymes or reduced

availability of essential endogenous cofactors.

Sex-dependent variations in drug metabolism have been well documented in rats but not in other

rodents. Young adult male rats metabolize drugs much faster than mature female rats or prepubertal

male rats. These differences in drug metabolism have been clearly associated with androgenic

hormones. Clinical reports suggest that similar sex-dependent differences in drug metabolism also

exist in humans for ethanol, propranolol, some benzodiazepines, estrogens, and salicylates.

Drug-Drug Interactions during Metabolism

Many substrates, by virtue of their relatively high lipophilicity, are retained not only at the active

site of the enzyme but remain nonspecifically bound to the lipid membrane of the endoplasmic

reticulum. In this state, they may induce microsomal enzymes; depending on the residual drug

levels at the active site, they also may competitively inhibit metabolism of a simultaneously

administered drug.

Enzyme-inducing drugs include various sedative-hypnotics, tranquilizers, anticonvulsants, and

insecticides (Table 4–5). Patients who routinely ingest barbiturates, other sedative-hypnotics, or

tranquilizers may require considerably higher doses of warfarin (an oral anticoagulant) to maintain

a prolonged prothrombin time. On the other hand, discontinuance of the sedative may result in

reduced metabolism of the anticoagulant and bleeding—a toxic effect of the ensuing enhanced

plasma levels of the anticoagulant. Similar interactions have been observed in individuals receiving

various combination drug regimens such as antipsychotics or sedatives with contraceptive agents,

sedatives with anticonvulsant drugs, and even alcohol with hypoglycemic drugs (tolbutamide).

Table 4–5. Partial List of Drugs That Enhance Drug Metabolism in Humans.

Inducer Drug Whose Metabolism Is Enhanced

Benzo[a]pyrene Theophylline

Chlorcyclizine Steroid hormones

Ethchlorvynol Warfarin

Glutethimide Antipyrine, glutethimide, warfarin

Griseofulvin Warfarin

Phenobarbital and

other barbiturates

Barbiturates, chloramphenicol, chlorpromazine, cortisol, coumarin

anticoagulants, desmethylimipramine, digitoxin, doxorubicin, estradiol,

phenylbutazone, phenytoin, quinine, testosterone

Phenylbutazone Aminopyrine, cortisol, digitoxin

Phenytoin Cortisol, dexamethasone, digitoxin, theophylline

Rifampin Coumarin anticoagulants, digitoxin, glucocorticoids,methadone,

metoprolol, oral contraceptives, prednisone, propranolol, quinidine

Secobarbital is an exception. See Table 4–6 and text.

It must also be noted that an inducer may enhance not only the metabolism of other drugs but also

its own metabolism. Thus, continued use of some drugs may result in a pharmacokinetic type of

tolerance—progressively reduced effectiveness due to enhancement of their own metabolism.

Conversely, simultaneous administration of two or more drugs may result in impaired elimination

of the more slowly metabolized drug and prolongation or potentiation of its pharmacologic effects

(Table 4–6). Both competitive substrate inhibition and irreversible substrate-mediated enzyme

inactivation may augment plasma drug levels and lead to toxic effects from drugs with narrow

therapeutic indices. Similarly, allopurinol both prolongs the duration and enhances the

chemotherapeutic action of mercaptopurine by competitive inhibition of xanthine oxidase.

Consequently, to avoid bone marrow toxicity, the dose of mercaptopurine is usually reduced in

patients receiving allopurinol. Cimetidine, a drug used in the treatment of peptic ulcer, has been

shown to potentiate the pharmacologic actions of anticoagulants and sedatives. The metabolism of

the sedative chlordiazepoxide has been shown to be inhibited by 63% after a single dose of

cimetidine; such effects are reversed within 48 hours after withdrawal of cimetidine.

Table 4–6. Partial List of Drugs That Inhibit Drug Metabolism in Humans.

Inhibitor Drug Whose Metabolism Is Inhibited

Allopurinol, chloramphenicol,

isoniazid

Antipyrine, dicumarol,probenecid, tolbutamide

Cimetidine Chlordiazepoxide, diazepam, warfarin, others

Dicumarol Phenytoin

Diethylpentenamide Diethylpentenamide

Disulfiram Antipyrine, ethanol, phenytoin, warfarin

Ethanol Chlordiazepoxide (?), diazepam (?), methanol

Grapefruit juice

Alprazolam, atorvastatin,cisapride, cyclosporine, midazolam,

triazolam

Ketoconazole Cyclosporine, astemizole, terfenadine

Nortriptyline Antipyrine

Oral contraceptives Antipyrine

Phenylbutazone Phenytoin, tolbutamide

Secobarbital Secobarbital

Troleandomycin Theophylline, methylprednisolone

Active components in grapefruit juice include furanocou-marins such as 6´, 7´-

dihydroxybergamottin (which is known to inactivate both intestinal and liver CYP3A4) as well as

other unknown components that inhibit P-glycoprotein-mediated intestinal drug efflux and

consequently further enhance the bioavailability of certain drugs such as cyclosporine.

Impaired metabolism may also result if a simultaneously administered drug irreversibly inactivates

a common metabolizing enzyme. These inhibitors, in the course of their metabolism by cytochrome

P450, inactivate the enzyme and result in impairment of their own metabolism and that of other

cosubstrates.

Interactions between Drugs & Endogenous Compounds

Various drugs require conjugation with endogenous substrates such as glutathione, glucuronic acid,

and sulfate for their inactivation. Consequently, different drugs may compete for the same

endogenous substrates, and the faster-reacting drug may effectively deplete endogenous substrate

levels and impair the metabolism of the slower-reacting drug. If the latter has a steep dose-response

curve or a narrow margin of safety, potentiation of its pharmacologic and toxic effects may result.

Diseases Affecting Drug Metabolism

Acute or chronic diseases that affect liver architecture or function markedly affect hepatic

metabolism of some drugs. Such conditions include alcoholic hepatitis, active or inactive alcoholic

cirrhosis, hemochromatosis, chronic active hepatitis, biliary cirrhosis, and acute viral or drug-

induced hepatitis. Depending on their severity, these conditions may significantly impair hepatic

drug-metabolizing enzymes, particularly microsomal oxidases, and thereby markedly affect drug

elimination. For example, the half-lives of chlordiazepoxide and diazepam in patients with liver

cirrhosis or acute viral hepatitis are greatly increased, with a corresponding prolongation of their

effects. Consequently, these drugs may cause coma in patients with liver disease when given in

ordinary doses.

Some drugs are metabolized so readily that even marked reduction in liver function does not

significantly prolong their action. However, cardiac disease, by limiting blood flow to the liver, may

impair disposition of those drugs whose metabolism is flow-limited (Table 4–7). These drugs are so

readily metabolized by the liver that hepatic clearance is essentially equal to liver blood flow.

Pulmonary disease may also affect drug metabolism as indicated by the impaired hydrolysis of

procainamide and procaine in patients with chronic respiratory insufficiency and the increased half-

life of antipyrine in patients with lung cancer. Impairment of enzyme activity or defective formation

of enzymes associated with heavy metal poisoning or porphyria also results in reduction of hepatic

drug metabolism.

Table 4–7. Rapidly Metabolized Drugs Whose Hepatic Clearance Is Blood Flow-Limited.

Alprenolol

Amitriptyline

Clomethiazole

Desipramine

Imipramine

Isoniazid

Labetalol

Lidocaine

Meperidine

Morphine

Pentazocine

Propoxyphene

Propranolol

Verapamil

Although the effects of endocrine dysfunction on drug metabolism have been well-explored in

experimental animal models, corresponding data for humans with endocrine disorders are scanty.

Thyroid dysfunction has been associated with altered metabolism of some drugs and of some

endogenous compounds as well. Hypothyroidism increases the half-life of antipyrine, digoxin,

methimazole, and practolol, whereas hyperthyroidism has the opposite effect. A few clinical studies

in diabetic patients indicate no apparent impairment of drug metabolism although impairment has

been noted in diabetic rats. Malfunctions of the pituitary, adrenal cortex, and gonads markedly

impair hepatic drug metabolism in rats. On the basis of these findings, it may be supposed that such

disorders could significantly affect drug metabolism in humans. However, until sufficient evidence

is obtained from clinical studies in patients, such extrapolations must be considered tentative.

Chapter 5. Basic & Clinical Evaluation of New Drugs

Basic & Clinical Evaluation of New Drugs: Introduction

Acknowledgment: I thank Wallace Dairman for comments and Kevin Abbey and Sema Ashkouri

for their research and administrative efforts.

New drug developments have revolutionized the practice of medicine, converting many once fatal

or debilitating diseases into almost routine therapeutic exercises. For example, deaths from

cardiovascular disease and stroke have decreased by more than 50% in the USA over the past 30

years. This decline is due—in part—to the discovery and increased use of antihypertensives,

cholesterol synthesis inhibitors, and drugs that prevent or dissolve blood clots. The process of drug

discovery and development has been greatly affected by investment in new technology and by

governmental support of medical research.

In most countries, the testing of therapeutic agents is now regulated by legislation and closely

monitored by governmental agencies. This chapter summarizes the process by which new drugs are

discovered, developed, and regulated. While the examples used reflect the experience in the USA,

the pathway of new drug development is generally the same worldwide.

One of the first steps in the development of a new drug is the discovery or synthesis of a potential

new drug molecule and correlating this molecule with an appropriate biologic target. Repeated

application of this approach leads to compounds with increased potency and selectivity (Figure 5–

1). By law, the safety and efficacy of drugs must be defined before they can be marketed. In

addition to in vitro studies, most of the biologic effects of the molecule must be characterized in

animals before human drug trials can be started. Human testing must then go forward in three

conventional phases before the drug can be considered for approval for general use. A fourth phase

of data gathering and safety monitoring follows after approval for general use.

Figure 5–1.

The development and testing process required to bring a drug to market in the USA. Some of the

requirements may be different for drugs used in life-threatening diseases.

Enormous costs, from $150 million to over $800 million, are involved in the research and

development of a single successful new drug. Thousands of compounds may be synthesized and

hundreds of thousands tested from existing libraries of compounds for each successful new drug

that reaches the market. It is primarily because of the economic investment and risks involved as

well as the need for multiple interdisciplinary technologies that most new drugs are developed in

pharmaceutical companies. At the same time, the incentives to succeed in drug development are

equally enormous. The worldwide market for ethical (prescription) pharmaceuticals in 2001 was

$364 billion. Moreover, it has been estimated that during the second half of the 20th century,

medications produced by the pharmaceutical industry saved more than 1.5 million lives and $140

billion in the costs of treatment for tuberculosis, poliomyelitis, coronary artery disease, and

cerebrovascular disease alone. In the USA, approximately 10% of the health care dollar is presently

spent on prescription drugs.

Drug Discovery

Most new drug candidates are launched through one or more of five approaches:

1. Identification or elucidation of a new drug target

2. Rational drug design based on an understanding of biologic mechanisms, drug receptor

structure, and drug structure

3. Chemical modification of a known molecule

4. Screening for biologic activity of large numbers of natural products; banks of previously

discovered chemical entities; and large libraries of peptides, nucleic acids, and other organic

molecules

5. Biotechnology and cloning using genes to produce larger peptides and proteins.

Moreover, automation, miniaturization and informatics have facilitated the process known

as "high through-put screening," which permits millions of assays per month.

Major attention is now being given to the discovery of entirely new targets for drug therapy. These

targets are emerging from studies with genomics, proteomics, and molecular pharmacology and are

expected to increase the number of useful biologic or disease targets ten-fold and thus be a positive

driver for new and improved drugs.

Drug Screening

Regardless of the source or the key idea leading to a drug candidate molecule, testing it involves a

sequence of experimentation and characterization called drug screening. A variety of biologic

assays at the molecular, cellular, organ system, and whole animal levels are used to define the

activity and selectivity of the drug. The type and number of initial screening tests depend on the

pharmacologic goal. Anti-infective drugs will generally be tested first against a variety of infectious

organisms, hypoglycemic drugs for their ability to lower blood sugar, etc. In addition, the molecule

will also be studied for a broad array of other actions to establish the mechanism of action and

selectivity of the drug. This has the advantage of demonstrating unsuspected toxic effects and

occasionally discloses a previously unsuspected therapeutic action. The selection of molecules for

further study is most efficiently conducted in animal models of human disease. Where good

predictive models exist (eg, hypertension or thrombotic disease), we generally have adequate drugs.

Good drugs are conspicuously lacking for diseases for which models are poor or not yet available,

eg, Alzheimer's disease.

Some of the studies performed during drug screening are listed in Table 5–1 and define the

pharmacologic profile of the drug. For example, a broad range of tests would be performed on a

drug designed to act as an antagonist at vascular -adrenoceptors for the treatment of hypertension.

At the molecular level, the compound would be screened for receptor binding affinity to cell

membranes containing receptors, possibly cloned human receptors, other receptors, and binding

sites on enzymes. Early studies would be done on liver cytochrome P450 enzymes to determine

whether the molecule of interest is likely to be a substrate or inhibitor of these enzymes.

Table 5–1. Pharmacologic Profile Tests.

Experimental Method or

Target Organ

Species or Tissue Route of

Administration

Measurement

Molecular

Receptor binding

(example: -

adrenoceptors)

Cell membrane

fractions from

organs or cultured

cells; cloned

receptors

In vitro Receptor affinity and

selectivity

Enzyme activity

(examples: tyrosine

Sympathetic nerves;

adrenal glands;

In vitro Enzyme inhibition and

selectivity

hydroxylase, dopamine-3-

hydroxylase, monoamine

oxidase)

purified enzymes

Cytochrome P450 Liver In vitro Enzyme inhibition; effects on

drug metabolism

Cellular

Cell function Cultured cells In vitro Evidence for receptor

activity—agonism or

antagonism (example: effects

on cyclic nucleotides)

Isolated tissue Blood vessels, heart,

lung, ileum (rat or

guinea pig)

In vitro Effects on vascular contraction

and relaxation; selectivity for

vascular receptors; effects on

other smooth muscles

Systems/disease models

Dog, cat

(anesthetized)

Parenteral Systolic-diastolic changes Blood pressure

Rat, hypertensive

(conscious)

Oral Antihypertensive effects

Dog (conscious) Oral Electrocardiography Cardiac effects

Dog (anesthetized) Parenteral Inotropic, chronotropic effects,

cardiac output, total peripheral

resistance

Peripheral autonomic

nervous system

Dog (anesthetized) Parenteral Effects on response to known

drugs and electrical

stimulation of central and

peripheral autonomic nerves

Respiratory effects Dog, guinea pig Parenteral Effects on respiratory rate and

amplitude, bronchial tone

Diuretic activity Dog Oral, parenteral Natriuresis, kaliuresis, water

diuresis, renal blood flow,

glomerular filtration rate

Gastrointestinal effects Rat Oral Gastrointestinal motility and

secretions

Circulating hormones,

cholesterol, blood sugar

Rat, dog Parenteral, oral Serum concentration

Blood coagulation Rabbit Oral Coagulation time, clot

retraction, prothrombin time

Central nervous system Mouse, rat Parenteral, oral Degree of sedation, muscle

relaxation, locomotor activity,

stimulation

Effects on cell function would be studied to determine the efficacy of the compound. Evidence

would be obtained about whether the drug is an agonist, partial agonist, or antagonist at receptors.